Chromatographic materials for the separation of unsaturated molecules

ABSTRACT

The present disclosure relates to a method of separating a compound of interest, particularly unsaturated compound(s) of interest, from a mixture. The compound is separated using a column having a chromatographic stationary phase material for various different modes of chromatography containing a first substituent and a second substituent. The first substituent minimizes compound retention variation over time under chromatographic conditions. The second substituent chromatographically and selectively retains the compound by incorporating one or more aromatic, polyaromatic, heterocyclic aromatic, or polyheterocyclic aromatic hydrocarbon groups, each group being optionally substituted with an aliphatic group. In some examples, the present disclosure can include a chromatographic system having a chromatographic column having a stationary phase with a chromatographic substrate containing silica, metal oxide, an inorganic-organic hybrid material, a group of block copolymers, or a combination thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/194,686, filed Mar. 1, 2014, which is a continuation-in-part ofInternational Patent Application Nos. PCT/US2013/041221, filed May 15,2013, and PCT/US2013/041207, filed May 15, 2013, which claim priority toProvisional Application No. 61/647,303, filed May 15, 2012, eachapplication of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present disclosure relates generally to chromatographic materialsfor the separation of unsaturated molecules. The present disclosurerelates more particularly, in various embodiments, to chromatographicmaterials for normal phase chromatography, high-pressure liquidchromatography, solvated gas chromatography, supercritical fluidchromatography, sub-critical fluid chromatography, carbon dioxide basedchromatography, hydrophilic interaction liquid chromatography andhydrophobic interaction liquid chromatography that mitigate or avoidretention drift or change while exhibiting useful overall retention forthe separation of unsaturated molecules, as well as correspondingapparatuses, kits, methods of manufacture, and methods of use.

BACKGROUND OF THE INVENTION

Chromatography is the collective term for a set of laboratory techniquesfor the separation of mixtures. The mixture is dissolved in a mobilephase, which carries it through a stationary phase. The variousconstituents of the mixture travel at different speeds, causing them toseparate. The separation is based on differential partitioning betweenthe mobile and stationary phases. Subtle differences in a compound'spartition coefficient result in differential retention on the stationaryphase, thus changing the separation. Chromatography is useful for theseparation of compounds that are structurally related, such asregio-isomers, chiral, diastereomers, etc. Some techniques, includingSFC, are known for being particularly useful for separating structurallyrelated vitamins, natural products and chemical materials. Often,however, chromatographic techniques are insufficient to separate allstructurally related compounds. For example, critical pairs of relatedvitamins (e.g., D2 and D3, K1 and K2) are difficult to separate/resolve.

Packing materials for fluid or liquid chromatography can be generallyclassified into two types: organic materials (e.g., polydivinylbenzene)and inorganic materials (e.g., silica). Many organic materials arechemically stable against strongly alkaline and strongly acidic mobilephases, allowing flexibility in the choice of mobile phase compositionand pH. However, organic chromatographic materials can result in columnswith low efficiency, particularly with low molecular-weight analytes.Many organic chromatographic materials not only lack the mechanicalstrength of typical chromatographic silica and also shrink and swellwhen the composition of the mobile phase is changed.

Silica is widely used in High Performance Liquid Chromatography (HPLC),Ultra High Performance Liquid Chromatography (UHPLC), and SupercriticalFluid Chromatography (SFC). Some applications employ silica that hasbeen surface-derivatized with an organic functional group such asoctadecyl (C18), octyl (C8), phenyl, amino, cyano, and the like. Asstationary phases for HPLC, these packing materials can result incolumns that have high efficiency and do not show evidence of shrinkingor swelling.

Hybrid materials can provide solutions to certain chromatographicproblems experienced with silica based packing materials. Hybridmaterials can provide improvements including improved high and low pHstability, mechanical stability, peak shape when used at pH 7,efficiency, retentivity, and desirable chromatographic selectivity.

However, potential problems can exist for conventional hybrid materialsand silica materials in other applications. One problem is poor peakshape for bases when used at low pH, which can negatively impactloadability and peak capacity when used at low pH. Another problem is achange in acidic and basic analyte retention times (denoted ‘drift’)after a column is exposed to repeated changes in mobile phase pH (e.g.,switching repeatedly from pH 10 to 3).

Another problem is retention drift or change, for example inchromatography modes with little water (e.g., less than 5%, less than1%). For example, retention drift or change is observed under standardSFC conditions for both silica and organic-inorganic hybrid (e.g., BEHTechnology™ materials available from Waters Technologies Corporation,Milford Mass.) based chromatographic phases, bonded and unbonded. OtherSFC stationary phases can also exhibit similar retention drift orchange.

SUMMARY OF THE INVENTION

In various aspects and embodiments, the present disclosure provideschromatographic materials for normal phase chromatography, high-pressureliquid chromatography, solvated gas chromatography, supercritical fluidchromatography, sub-critical fluid chromatography, carbon dioxide basedchromatography, hydrophilic interaction liquid chromatography andhydrophobic interaction liquid chromatography that mitigate or avoidretention drift or change while exhibiting useful overall retention forthe separation of unsaturated molecules, as well as correspondingapparatuses, kits, methods of manufacture, and methods of use.

The present disclosure includes various additional advantages, includingbut not limited to, the ability to selection/design selectivity throughselection/design of the chemical modifications.

In one embodiment, the present disclosure relates to a method ofseparating a compound of interest from a mixture, the method comprisingproviding a mixture containing the compound of interest, introducing aportion of the mixture to a chromatographic system having achromatographic column, and eluting the separated compound of interestfrom the column, wherein the column has a stationary phase having thefollowing structure (i):[X](W)_(a)(Q)_(b)(T)_(c)  (i)

wherein X is a chromatographic substrate containing silica, metal oxide,an inorganic-organic hybrid material, a group of block copolymers, orcombinations thereof, W is selected from the group consisting ofhydrogen and hydroxyl, wherein W is bound to the surface of X, Q is afirst substituent which minimizes analyte retention variation over timeunder chromatographic conditions having low water concentrations, T is asecond substituent which chromatographically retains the analyte,wherein T has one or more aromatic, polyaromatic, heterocyclic aromatic,or polyheterocyclic aromatic hydrocarbon groups, each group beingoptionally substituted with an aliphatic group; and b and c are positivenumbers, 0.05≤(b/c)≤100, and a ≥0.

In some embodiments, Q has the following structure (ii):

wherein n1 is an integer from 1-30, n2 is an integer from 1-30, R¹, R²,R³ and R⁴ are each independently selected from the group consisting ofhydrogen, hydroxyl, fluoro, methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, tert-butyl, lower alkyl, a protected or deprotectedalcohol, and a zwitterion, Z is either (a) a surface attachment grouphaving the formula (B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si—, wherein x is an integerfrom 1-3, y is an integer from 0-2, z is an integer from 0-2, andx+y+z=3, R⁵ and R⁶ are each independently selected from the groupconsisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,tert-butyl, substituted or unsubstituted aryl, cyclic alkyl, branchedalkyl, lower alkyl, a protected or deprotected alcohol, a zwitteriongroup and a siloxane bond, and B¹ is a siloxane bond, or (b) anattachment to a surface organofunctional hybrid group through a directcarbon-carbon bond formation or through a heteroatom, ester, ether,thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle,triazole, or urethane linkage, or (c) an adsorbed, surface group that isnot covalently attached to the surface of the material, Y is an embeddedpolar functionality, a bond or an aliphatic group, and A is selectedfrom the group consisting of an hydrophilic terminal group, afunctionizable group, hydrogen, hydroxyl, fluoro, methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, a lower alkyl anda polarizable group.

In some embodiments, T has the following structure (iii):

wherein m¹ is an integer from 1-30, m² is an integer from 1-30, m³ is aninteger from 1-3, R⁷, R⁸, R⁹ and R¹⁰ are each independently selectedfrom the group consisting of hydrogen, hydroxyl, fluoro, methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, lower alkyl, aprotected or deprotected alcohol, a zwitterion, an aromatic hydrocarbongroup and a heterocyclic aromatic hydrocarbon group, Z is (a) a surfaceattachment group having the formula (B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si—, whereinx is an integer from 1-3, y is an integer from 0-2, z is an integer from0-2, and x+y+z=3, R⁵ and R⁶ are each independently selected from thegroup consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, tert-butyl, substituted or unsubstituted aryl, cyclic alkyl,branched alkyl, lower alkyl, a protected or deprotected alcohol, azwitterion group and a siloxane bond, and B¹ is a siloxane bond, (b) anattachment to a surface organofunctional hybrid group through a directcarbon-carbon bond formation or through a heteroatom, ester, ether,thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle,triazole, or urethane linkage, or (c) an adsorbed, surface group that isnot covalently attached to the surface of the material, Y is an embeddedpolar functionality, a bond or an aliphatic group, D is selected fromthe group consisting of a bond, N, O, S, —(CH₂)₀₋₁₂—N—R¹¹R¹²,—(CH₂)₀₋₁₂—O—R¹¹, —(CH₂)₀₋₁₂—S—R, —(CH₂)₀₋₁₂—N—(CH₂)₀₋₁₂—R¹¹R¹²,—(CH₂)₀₋₁₂—O—(CH₂)₀₋₁₂—R¹¹, —(CH₂)₀₋₁₂—S—(CH₂)₀₋₁₂—R¹¹,—(CH₂)₀₋₁₂—S(O)₁₋₂—(CH₂)₀₋₁₂—N—R¹¹R¹²,—(CH₂)₀₋₁₂—S(O)₁₋₂—(CH₂)₀₋₁₂—O—R¹¹, —(CH₂)₀₋₁₂—S(O)₁₋₂—(CH₂)₀₋₁₂—S—R¹¹;—(CH₂)₀₋₁₂—S(O)₁₋₂—(CH₂)₀₋₁₂—N—(CH₂)₀₋₁₂—R¹¹R¹²,—(CH₂)₀₋₁₂—S(O)₁₋₂—(CH₂)₀₋₁₂—O—(CH₂)₀₋₁₂—R, and—(CH₂)₀₋₁₂—S(O)₁₋₂—(CH₂)₀₋₁₂—S—(CH₂)₀₋₁₂—R¹¹, R¹¹ is a firstmono-aromatic, polyaromatic, heterocyclic aromatic, or polyheterocyclicaromatic group, R¹² is a hydrogen, an aliphatic group or a secondmono-aromatic, polyaromatic, heterocyclic aromatic, or polyheterocyclicaromatic group, wherein R¹¹ and R¹² are optionally substituted with analiphatic group. Various fractions of Q, T, or both can be polymerized.

In another embodiment, the present disclosure relates to method forseparating a lipid, vitamin or polycyclic aromatic hydrocarbon from amixture.

In another embodiment, the present disclosure relates to achromatographic stationary phase having the following structure (i):[X](W)_(a)(Q)_(b)(T)_(c)  (i)

wherein X is a chromatographic substrate containing silica, metal oxide,an inorganic-organic hybrid material, a group of block copolymers, orcombinations thereof, W is selected from the group consisting ofhydrogen and hydroxyl, wherein W is bound to the surface of X, Q is afirst substituent which minimizes analyte retention variation over timeunder chromatographic conditions having low water concentrations, T is asecond substituent which chromatographically retains the analyte,wherein T has one or more mono-aromatic, polyaromatic, heterocyclicaromatic, or polyheterocyclic aromatic groups, each group beingoptionally substituted with an aliphatic group; and b and c are positivenumbers, 0.05≤(b/c)≤100, and a ≥0.

In another embodiment, the present disclosure relates to a column,capillary column, monolithic column, microfluidic device or apparatusfor normal phase chromatography, high-pressure liquid chromatography,solvated gas chromatography, supercritical fluid chromatography,sub-critical fluid chromatography, carbon dioxide based chromatography,hydrophilic interaction liquid chromatography or hydrophobic interactionliquid chromatography comprising a housing having at least one walldefining a chamber having an entrance and an exit, and a stationaryphase having the above structure, i.e. (i), disposed therein, whereinthe housing and stationary phase are adapted for normal phasechromatography, high-pressure liquid chromatography, solvated gaschromatography, supercritical fluid chromatography, sub-critical fluidchromatography, carbon dioxide based chromatography, hydrophilicinteraction liquid chromatography or hydrophobic interaction liquidchromatography.

In another embodiment, the present disclosure relates to a kit fornormal phase chromatography, high-pressure liquid chromatography,solvated gas chromatography, supercritical fluid chromatography,sub-critical fluid chromatography, carbon dioxide based chromatography,hydrophilic interaction liquid chromatography or hydrophobic interactionliquid chromatography comprising a housing having at least one walldefining a chamber having an entrance and an exit, and a stationaryphase having the above structure, i.e. (i), disposed therein, whereinthe housing and stationary phase are adapted for normal phasechromatography, high-pressure liquid chromatography, solvated gaschromatography, supercritical fluid chromatography, sub-critical fluidchromatography, carbon dioxide based chromatography, hydrophilicinteraction liquid chromatography or hydrophobic interaction liquidchromatography; and instructions for performing normal phasechromatography, high-pressure liquid chromatography, solvated gaschromatography, supercritical fluid chromatography, sub-critical fluidchromatography, carbon dioxide based chromatography, hydrophilicinteraction liquid chromatography or hydrophobic interaction liquidchromatography with the housing and stationary phase.

In another embodiment, the present disclosure relates to a method forpreparing a stationary phase having the above structure, i.e. (i),comprising reacting a chromatographic substrate with a silane couplingagent having a pendant reactive group, reacting a second chemical agentcomprising one or more aromatic, polyaromatic, heterocyclic aromatic, orpolyheterocyclic aromatic hydrocarbon groups with the pendant reactivegroup; and neutralize any remaining unreacted pendant reactive groups,thereby producing the stationary phase.

In another embodiment, the present disclosure relates to a method forpreparing a stationary phase having the above structure, i.e. (i),comprising oligomerizing a silane coupling agent having a pendantreactive group, reacting a core surface with the oligomerized silanecoupling agent, reacting a second chemical agent comprising one or morearomatic, polyaromatic, heterocyclic aromatic, or polyheterocyclicaromatic hydrocarbon groups with the pendant reactive group; andneutralize any remaining unreacted pendant reactive groups, therebyproducing the stationary phase.

In another embodiment, the present disclosure relates to a method formitigating or preventing retention drift in normal phase chromatography,high-pressure liquid chromatography, solvated gas chromatography,supercritical fluid chromatography, sub-critical fluid chromatography,carbon dioxide based chromatography, hydrophilic interaction liquidchromatography or hydrophobic interaction liquid chromatographycomprising chromatographically separating a sample using achromatographic device comprising a chromatographic stationary phasehaving the above structure, i.e. (i), disposed therein, therebymitigating or preventing retention drift.

The present disclosure advantageously mitigates or avoids retentiondrift or change while exhibiting useful overall retention, particularlyfor unsaturated molecules or compounds of interest. For example, in SFC,retention drift or change can be (among various other theories)attributed to alkoxylation of solvent accessible silanols on theparticle under the standard CO₂/MeOH mobile phase (and/or by otheralcohol co-solvents) utilized for SFC. This is a problem, as usersobserve a change in the chromatography (e.g., retention time) obtainedon their SFC system as the column ages, and again when a new,non-alkoxylated column is put on the system.

In various aspects and embodiments, the present disclosure providesvarious solutions to such retention drift or change and related problems(e.g., retention, peak shape, and the like) through selection and/ormodification of the chromatographic material. For example, the inventionincludes specialized functionalization of a chromatographic core surface(e.g., with particular functional groups, and combinations thereof),which essentially prevent chromatographic interaction between andanalyte and the chromatographic core surface, which maintaining desiredinteraction between the analyte and the chromatographic material.

In other various aspects and embodiments, the present disclosure relatesto chromatographic materials having greatly reduced secondaryinteractions with the base particle surface (e.g., unwantedinteractions, non-specific adsorption). Secondary interactions ofanalyte with the material surface can occur due to silanols, pendanthydrophobic groups and polymer or hybrid backbone chains.

In various aspects and embodiments, the present disclosure providesnumerous advantages. For example, the present disclosure can provide fora stationary phase capable of resolving analytes across all classes(e.g., acidic, basic and neutral), particularly unsaturated analytes,with superior retention, peak capacity and peak shape, peak shape forbases being of lesser importance. In various examples, the presentdisclosure can effectively masks silanols from the analytes of interestproducing a predictable and stable chromatographic separation. Invarious examples, the present disclosure can effectively eliminateretention drift or change due to unwanted support surface interactionswith analyte. The present disclosure can be especially effective atmasking silanols on silica or silica hybrid materials. In variousexamples, the present disclosure can improve peak capacity and tailingacross all analyte classes, but especially with bases. In variousexamples, the present disclosure can avoid pore clogging, despitebonding with oligomeric siloxanes (e.g., as compared to conventionalpolymeric coatings of porous silica materials, which can result inclogging of the pores greatly decreasing the available surface area ofthe materials and leading to inhomogeneous surfaces—despite thepromotion of oligomerization of silanes in the present disclosure thereis no evidence of pore clogging or decreased surface area).

The present disclosure provides advantages over the prior art based onits unique chemistry and performance properties. For example, the liquidseparations of unsaturated compounds usually involves separationsperformed on C18 bonded phases, such as ACQUITY UPC²® HSS C18 SB. Onthat material, alkyl chains are the retention selectors resulting inhigh methylene/hydrophobic selectivity, but little shape/isomericselectivity. The present disclosure provides retention selectors capableof both methylene/hydrophobic selectivity and shape/isomericselectivity. For example, stationary phases of the present disclosureare exceptional at retaining and separating fat-soluble vitamins, lipidsand metabolites as well as providing enhanced shape/isomeric selectivityas compared to C18 bonded phases.

The present disclosure is described in further detail by the drawingsand examples below, which are used only for illustration purposes andare not limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure can be more readily understood in the context ofthe following drawings and Detailed Description. It will be understoodby a practitioner of ordinary skill in the art that the followingdrawings are not necessarily to scale, emphasis instead being placed onillustrating the inventive concepts of the present invention.

FIGS. 1A and 1B show the structure of glycidoxypropyltrimethoxysilane(GPTMS) (FIG. 1A) and 1-aminoanthracene (FIG. 1B).

FIG. 2 shows a schematic of a reaction between an unmodifiedchromatographic surface and GPTMS.

FIG. 3 shows a schematic of a reaction between a modifiedchromatographic surface and 1-aminoanthracene.

FIG. 4 shows a schematic of a chromatographic surface crosslinked withthe modifying agents GPTMS and 1-aminoanthracene.

FIG. 5 shows a schematic of two potential synthetic routes to preparinga chromatographic stationary phase of the present disclosure.

FIG. 6 shows a graph of the percent retention of an analyte eluted usingunmodified BEH particles as a stationary phase, and BEH particlesmodified in accordance with the present disclosure.

FIGS. 7A and 7B show exemplary lipid separations using a1-aminoanthracene based stationary phase as described in Example 9.

FIG. 8A shows chromatograms of C22:0 and C20:0 using a 1-aminoanthracenebased stationary phase as described in Example 9.

FIG. 8B shows chromatograms of C16:0, C12:0 and C8:0 using a1-aminoanthracene based stationary phase as described in Example 9.

FIG. 9A shows chromatograms of C22:0 and C20:0 using a 1-aminoanthracenebased stationary phase as described in Example 9.

FIG. 9B shows chromatograms of C16:0, C12:0 and C8:0 using a1-aminoanthracene based stationary phase as described in Example 9.

FIG. 10A shows chromatograms of C24:1 and C22:1 using a1-aminoanthracene based stationary phase as described in Example 9.

FIG. 10B shows chromatograms of C20:1, C18:1 and C14:1 using a1-aminoanthracene based stationary phase as described in Example 9.

FIG. 11A shows chromatograms of C24:1 and C22:1 using a1-aminoanthracene based stationary phase as described in Example 9.

FIG. 11B shows chromatograms of C20:1, C18:1 and C14:1 using a1-aminoanthracene based stationary phase as described in Example 9.

FIG. 12A shows chromatograms of C18:0. C18:1 and C18:2 using a1-aminoanthracene based stationary phase as described in Example 9.

FIG. 12B shows chromatograms of C22:1. C22:2 and C22:6 using a1-aminoanthracene based stationary phase as described in Example 9.

FIG. 13A shows chromatograms of C18:0, C18:1 and C18:2 using a1-aminoanthracene based stationary phase as described in Example 9.

FIG. 13B shows chromatograms of C22:1, C22:2 and C22:6 using a1-aminoanthracene based stationary phase as described in Example 9.

FIGS. 14 and 15 show chromatograms of various linolenic andeicosadienoic acids using a 1-aminoanthracene based stationary phase asdescribed in Example 9.

FIG. 16 shows an exemplary lipid separation achieved using a1-aminoanthracene based stationary phase as described in Example 9.

FIG. 17A shows a chromatogram of various lipids using a1-aminoanthracene based stationary phase as described in Examples 3 and10.

FIG. 17B shows a chromatogram of various lipids using a 2-picolylaminebased stationary phase as described in Examples 3 and 10.

FIG. 17C shows a chromatogram of various lipids using a pyridine basedstationary phase as described in Examples 3 and 10.

FIG. 17D shows a chromatogram of various lipids using a 6-aminoquinolinebased stationary phase as described in Examples 3 and 10.

FIG. 17E shows a chromatogram of various lipids using an aniline basedstationary phase as described in Examples 3 and 10.

FIG. 17F shows a chromatogram of various lipids using a GPTMS basedstationary phase as described in Examples 3 and 10.

FIG. 17G shows a chromatogram of various lipids using a 4-n-octylanilinebased stationary phase as described in Examples 3 and 10.

FIG. 18A shows chromatograms of C18:0, C18:1 and C18:2 using a1-aminoanthracene based stationary phase as described in Examples 3 and10.

FIG. 18B shows chromatograms of C22:1, C22:2 and C22:6 using a1-aminoanthracene based stationary phase as described in Examples 3 and10.

FIG. 19A shows chromatograms of C18:0, C18:1 and C18:2 using a2-picolylamine based stationary phase as described in Examples 3 and 10.

FIG. 19B shows chromatograms of C22:1, C22:2 and C22:6 using a2-picolylamine based stationary phase as described in Examples 3 and 10.

FIG. 20A shows chromatograms of C18:0, C18:1 and C18:2 using a pyridinebased stationary phase as described in Examples 3 and 10.

FIG. 20B shows chromatograms of C22:1, C22:2 and C22:6 using a pyridinebased stationary phase as described in Examples 3 and 10.

FIG. 21A shows chromatograms of C18:0, C18:1 and C18:2 using a6-aminoquinoline based stationary phase as described in Examples 3 and10.

FIG. 21B shows chromatograms of C22:1, C22:2 and C22:6 using a6-aminoquinoline based stationary phase as described in Examples 3 and10.

FIG. 22A shows chromatograms of C18:0, C18:1 and C18:2 using a anilinebased stationary phase as described in Examples 3 and 10.

FIG. 22B shows chromatograms of C22:1, C22:2 and C22:6 using a anilinebased stationary phase as described in Examples 3 and 10.

FIG. 23A shows chromatograms of C18:0, C18:1 and C18:2 using a GPTMSbased stationary phase as described in Examples 3 and 10.

FIG. 23B shows chromatograms of C22:1, C22:2 and C22:6 using a GPTMSbased stationary phase as described in Examples 3 and 10.

FIGS. 24A-24F show chromatograms of C18:0, C18:1, C18:2, C22:1, C22:2and C22:6 using a 4-n-octylaniline based stationary phase as describedin Examples 3 and 10

DETAILED DESCRIPTION OF THE INVENTION

In various aspects and embodiments, the present disclosure provideschromatographic materials for normal phase chromatography, high-pressureliquid chromatography, solvated gas chromatography, supercritical fluidchromatography, sub-critical fluid chromatography, carbon dioxide basedchromatography, hydrophilic interaction liquid chromatography andhydrophobic interaction liquid chromatography that mitigate or avoidretention drift or change while exhibiting useful overall retention forthe separation of unsaturated molecules, as well as correspondingapparatuses, kits, methods of manufacture, and methods of use. In someembodiments, the present disclosure also provides retention andseparation of structurally related compounds, such as critical pairswhich are difficult to separate.

The present disclosure advantageously mitigates or avoids retentiondrift or change while exhibiting useful overall retention. For example,in SFC, retention drift or change can be (among various other theories)attributed to alkoxylation of solvent accessible silanols on theparticle under the standard CO₂/MeOH mobile phase (and/or by otheralcohol co-solvents) utilized for SFC. This is a problem, as usersobserve a change in the chromatography (e.g., retention time) obtainedon their SFC system as the column ages, and again when a new,non-alkoxylated column is put on the system.

In various aspects and embodiments, the present disclosure providesvarious solutions to such retention drift or change and related problems(e.g., retention, peak shape, and the like) through selectivemodification of chromatographic materials and/or resolution of mixturesof unsaturated compounds of interest.

Definitions

In various aspects and embodiments, the present invention provides formitigating or preventing retention drift or change. “Retention drift” or“retention change” can include an undesired difference in elution timebetween chromatographic runs or experiments (e.g., in run 1, peak xelutes at time y, but in run 1+n, peak x elutes at time z). Thus,retention drift or change can result in undesired effects includingexperimental noise, irreproducibility, or failure. Accordingly, in abroad sense, mitigating or preventing retention drift or change includesaddressing or counteracting an undesired difference in elution timesbetween chromatographic runs, to the extent that the chromatographicexperiment provides and chromatographically acceptable result.

In some embodiments, mitigating or preventing retention drift or changeis not constant an absolute or constant value. For example, the amountof retention drift or change the can occur while still achieving achromatographically acceptable result can vary depending upon theacceptable error or variance in a given experiment, the complexity of asample (e.g., number and/or separation of peaks). The amount ofretention drift or change the can occur while still achieving achromatographically acceptable result can vary depending upon theduration or required reproducibility a given experiment (e.g., ifreproducibility is required over a greater number of runs, the allowableretention drift or change between runs can be smaller). Therefore, itshould be clear that mitigating or preventing retention drift or changedoes not necessarily mean the absolute elimination of retention drift orchange.

In some embodiments, mitigating or preventing retention drift or changecan be quantified. For example, retention drift or change can bemeasured for a single peak, or averaged over a set of peaks. Retentiondrift or change can be measured over a given period of time or number ofruns. Retention drift or change can be measured relative to a standardvalue, starting value, or between two or more given runs.

Furthermore, retention drift or change can be quantified by astandardized test. For example, the Average % Retention Change can becalculated by taking the percent difference of the average absolute peakretentions measured from the day 3, 10 or 30 chromatographic tests fromthe average absolute peak retentions measured on the day onechromatographic test. For each day tested, the columns can beequilibrated under one set of test conditions followed by multipleinjections of a first test mix and then equilibrated under a second setof condition followed by multiple injections of a second test mix.

In accordance with this standardized test, mitigating or preventingretention drift or change can comprises a retention drift or change of≤5% over 30 days, ≤4% over 30 days, ≤3% over 30 days, ≤2% over 30 days,≤5% over 30 days, ≤5% over 10 days, ≤4% over 10 days, ≤3% over 10 days,≤2% over 10 days, ≤1% over 10 days, ≤5% over 3 days, ≤4% over 3 days,≤3% over 3 days, ≤2% over 3 days, ≤1% over 3 days, ≤5% over 30 runs, ≤4%over 30 runs, ≤3% over 30 runs, ≤2% over 30 runs, ≤1% over 30 runs, ≤5%over 10 runs, ≤4% over 10 runs, ≤3% over 10 runs, ≤2% over 10 runs, ≤1%over 10 runs, ≤5% over 3 runs, ≤4% over 3 runs, ≤3% over 3 runs, ≤2%over 3 runs, or ≤1% over 3 runs.

In other embodiments, mitigating or preventing retention drift or changecan comprises a retention drift or change of ≤5.0, 4.9, 4.8, 4.7, 4.6,4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2,3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8,1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4,0.3, 0.2, or 0.1% over 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1days (or runs).

“High Purity” or “high purity chromatographic material” includes amaterial which is prepared from high purity precursors. In certainaspects, high purity materials have reduced metal contamination and/ornon-diminished chromatographic properties including, but not limited to,the acidity of surface silanols and the heterogeneity of the surface.

“Chromatographic surface” includes a surface which provides forchromatographic separation of a sample. In certain aspects, thechromatographic surface is porous. In some aspects, a chromatographicsurface can be the surface of a particle, a superficially porousmaterial or a monolith. In certain aspects, the chromatographic surfaceis composed of the surface of one or more particles, superficiallyporous materials or monoliths used in combination during achromatographic separation. In certain other aspects, thechromatographic surface is non-porous.

“Ionizable modifier” includes a functional group which bears an electrondonating or electron withdrawing group. In certain aspects, theionizable modifier contains one or more carboxylic acid groups, aminogroups, imido groups, amido groups, pyridyl groups, imidazolyl groups,ureido groups, thionyl-ureido groups or aminosilane groups, or acombination thereof. In other aspects, the ionizable modifier contains agroup bearing a nitrogen or phosphorous atom having a free electron lonepair. In certain aspects, the ionizable modifier is covalently attachedto the material surface and has an ionizable group. In some instances itis attached to the chromatographic material by chemical modification ofa surface hybrid group.

“Hydrophobic surface group” includes a surface group on thechromatographic surface which exhibits hydrophobicity. In certainaspects, a hydrophobic group can be a carbon bonded phase such as a C₄to C₁₈ bonded phase. In other aspects, a hydrophobic surface group cancontain an embedded polar group such that the external portion of thehydrophobic surface maintains hydrophobicity. In some instances it isattached to the chromatographic material by chemical modification of asurface hybrid group. In other instances the hydrophobic group can beC₄-C₃₀, embedded polar, chiral, phenylalkyl, or pentafluorophenylbonding and coatings.

“Chromatographic core” includes chromatographic materials, including butnot limited to an organic material such as silica or a hybrid material,as defined herein, in the form of a particle, a monolith or anothersuitable structure which forms an internal portion of the materials ofthe present disclosure. In certain aspects, the surface of thechromatographic core represents the chromatographic surface, as definedherein, or represents a material encased by a chromatographic surface,as defined herein. The chromatographic surface material can be disposedon or bonded to or annealed to the chromatographic core in such a waythat a discrete or distinct transition is discernible or can be bound tothe chromatographic core in such a way as to blend with the surface ofthe chromatographic core resulting in a gradation of materials and nodiscrete internal core surface. In certain embodiments, thechromatographic surface material can be the same or different from thematerial of the chromatographic core and can exhibit different physicalor physiochemical properties from the chromatographic core, including,but not limited to, pore volume, surface area, average pore diameter,carbon content or hydrolytic pH stability.

“Hybrid,” including “hybrid inorganic/organic material,” includesinorganic-based structures wherein an organic functionality is integralto both the internal or “skeletal” inorganic structure as well as thehybrid material surface. The inorganic portion of the hybrid materialcan be, e.g., alumina, silica, titanium, cerium, or zirconium or oxidesthereof, or ceramic material. “Hybrid” includes inorganic-basedstructures wherein an organic functionality is integral to both theinternal or “skeletal” inorganic structure as well as the hybridmaterial surface. As noted above, exemplary hybrid materials are shownin U.S. Pat. Nos. 4,017,528, 6,528,167, 6,686,035 and 7,175,913, thecontents of which are incorporated herein by reference in theirentirety.

The term “alicyclic group” includes closed ring structures of three ormore carbon atoms. Alicyclic groups include cycloparaffins or naphtheneswhich are saturated cyclic hydrocarbons, cycloolefins, which areunsaturated with two or more double bonds, and cycloacetylenes whichhave a triple bond. They do not include aromatic groups. Examples ofcycloparaffins include cyclopropane, cyclohexane and cyclopentane.Examples of cycloolefins include cyclopentadiene, cyclohexadiene andcyclooctatetraene. Alicyclic groups also include fused ring structuresand substituted alicyclic groups such as alkyl substituted alicyclicgroups. In the instance of the alicyclics such substituents can furthercomprise a lower alkyl, a lower alkenyl, a lower alkoxy, a loweralkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, ahydroxyl, —CF₃, —CN, or the like.

The term “aliphatic group” includes organic compounds characterized bystraight or branched chains, typically having between 1 and 24 carbonatoms. Aliphatic groups include alkyl groups, alkenyl groups and alkynylgroups. In complex structures, the chains can be branched orcross-linked. In some embodiments, the aliphatic group can includechains having between 2 and 24 carbon atoms, or 4 and 22 carbon atoms,or 6 and 20 carbon atoms, or 8 and 18 carbon atoms, or 10 and 16 carbonatoms, or 12 and 14 carbon atoms, or any combination of these numbers,such as between about 6 and 12 carbon atoms or 10 and 14 carbon atoms.Alkyl groups include saturated hydrocarbons having one or more carbonatoms, including straight-chain alkyl groups and branched-chain alkylgroups. Such hydrocarbon moieties can be substituted on one or morecarbons with, for example, a halogen, a hydroxyl, a thiol, an amino, analkoxy, an alkylcarboxy, an alkylthio, or a nitro group. Unless thenumber of carbons is otherwise specified, “lower aliphatic” as usedherein means an aliphatic group, as defined above (e.g., lower alkyl,lower alkenyl, lower alkynyl), but having from one to six carbon atoms.Representative of such lower aliphatic groups, e.g., lower alkyl groups,are methyl, ethyl, n-propyl, isopropyl, 2-chloropropyl, n-butyl,sec-butyl, 2-aminobutyl, isobutyl, tert-butyl, 3-thiopentyl and thelike. As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “thiol” means SH; and the term“hydroxyl” means —OH. Thus, the term “alkylamino” as used herein meansan alkyl group, as defined above, having an amino group attachedthereto. Suitable alkylamino groups include groups having 1 to about 12carbon atoms, preferably from 1 to about 6 carbon atoms. The term“alkylthio” refers to an alkyl group, as defined above, having asulfhydryl group attached thereto. Suitable alkylthio groups includegroups having 1 to about 12 carbon atoms, preferably from 1 to about 6carbon atoms. The term “alkylcarboxyl” as used herein means an alkylgroup, as defined above, having a carboxyl group attached thereto. Theterm “alkoxy” as used herein means an alkyl group, as defined above,having an oxygen atom attached thereto. Representative alkoxy groupsinclude groups having 1 to about 12 carbon atoms, preferably 1 to about6 carbon atoms, e.g., methoxy, ethoxy, propoxy, tert-butoxy and thelike. The terms “alkenyl” and “alkynyl” refer to unsaturated aliphaticgroups analogous to alkyls, but which contain at least one double ortriple bond respectively. Suitable alkenyl and alkynyl groups includegroups having 2 to about 12 carbon atoms, preferably from 1 to about 6carbon atoms.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups and cycloalkylsubstituted alkyl groups. In certain embodiments, a straight chain orbranched chain alkyl has 30 or fewer carbon atoms in its backbone, e.g.,C₁-C₃₀ for straight chain or C₃-C₃₀ for branched chain. In certainembodiments, a straight chain or branched chain alkyl has 20 or fewercarbon atoms in its backbone, e.g., C₁-C₂₀ for straight chain or C₃-C₂₀for branched chain, and more preferably 18 or fewer. Likewise, preferredcycloalkyls have from 4-10 carbon atoms in their ring structure and morepreferably have 4-7 carbon atoms in the ring structure. The term “loweralkyl” refers to alkyl groups having from 1 to 6 carbons in the chainand to cycloalkyls having from 3 to 6 carbons in the ring structure.

Moreover, the term “alkyl” (including “lower alkyl”) as used throughoutthe present disclosure includes both “unsubstituted alkyls” and“substituted alkyls,” the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino,arylamino, diarylamino and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. It willbe understood by those skilled in the art that the moieties substitutedon the hydrocarbon chain can themselves be substituted, if appropriate.Cycloalkyls can be further substituted, e.g., with the substituentsdescribed above. An “aralkyl” moiety is an alkyl substituted with anaryl, e.g., having 1 to 3 separate or fused rings and from 6 to about 18carbon ring atoms, e.g., phenylmethyl(benzyl).

The term “amino,” as used herein, refers to an unsubstituted orsubstituted moiety of the formula —NR_(a)R_(b), in which R_(a) and R_(b)are each independently hydrogen, alkyl, aryl, or heterocyclyl, or R_(a)and R_(b), taken together with the nitrogen atom to which they areattached, forms a cyclic moiety having from 3 to 8 atoms in the ring.Thus, the term “amino” includes cyclic amino moieties such aspiperidinyl or pyrrolidinyl groups, unless otherwise stated. An“amino-substituted amino group” refers to an amino group in which atleast one of R_(a) and R_(b), is further substituted with an aminogroup.

The term “aromatic group” includes unsaturated cyclic hydrocarbonscontaining one or more rings. The term “mono-aromatic” includesunsaturated cyclic hydrocarbons containing one ring. The term“polyaromatic” includes unsaturated cyclic hydrocarbons containing twoor more rings. Aromatic groups include 5- and 6-membered single-ringgroups which can include from zero to four heteroatoms, for example,furan, pyrrole, pyrroline, oxazole, thiazole, imidazole, imidazoline,pyrazole, pyrazoline, pyrazolidine, isoxazole, isothiazole, benzene,pyridine, pyridazine, pyrimidine, pyrazine, triazine, thiophene and thelike. The aromatic ring can be substituted at one or more ring positionswith, for example, a halogen, a lower alkyl, a lower alkenyl, a loweralkoxy, a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, anitro, a hydroxyl, —CF₃, —CN, or the like. Aromatic groups include 5-and 6-membered multiple-ring groups which can include from zero to eightheteroatoms, for example, indene, indolizine, indole, isoindole,indoline, indazole, benzimidazole, benzthiazole, naphthalene,quinolizine, quinoline, isoquinoline, cinnoline, phthalazine,quinazoline, quinoxaline, 1,8-naphthyridine, quinuclidine, fluorene,carbazole, anthracene, acridine, phanazine, phenothiazine, phenoxazine,pyrene, and the like. Polyaromatic groups include fused aromatic groups.

The term “aryl” includes 5- and 6-membered single-ring aromatic groupsthat can include from zero to four heteroatoms, for example,unsubstituted or substituted benzene, pyrrole, furan, thiophene,imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine,pyridazine and pyrimidine and the like. Aryl groups also includepolycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl andthe like. The aromatic ring can be substituted at one or more ringpositions with such substituents, e.g., as described above for alkylgroups. Suitable aryl groups include unsubstituted and substitutedphenyl groups. The term “aryloxy” as used herein means an aryl group, asdefined above, having an oxygen atom attached thereto. The term“aralkoxy” as used herein means an aralkyl group, as defined above,having an oxygen atom attached thereto. Suitable aralkoxy groups have 1to 3 separate or fused rings and from 6 to about 18 carbon ring atoms,e.g., O-benzyl.

The term “ceramic precursor” is intended include any compound thatresults in the formation of a ceramic material.

The term “chiral moiety” is intended to include any functionality thatallows for chiral or stereoselective syntheses. Chiral moieties include,but are not limited to, substituent groups having at least one chiralcenter, natural and unnatural amino-acids, peptides and proteins,derivatized cellulose, macrocyclic antibiotics, cyclodextrins, crownethers, and metal complexes.

The term “embedded polar functionality” is a functionality that providesan integral polar moiety such that the interaction with basic samplesdue to shielding of the unreacted silanol groups on the silica surfaceis reduced. Embedded polar functionalities include, but are not limitedto carbonate, amide, urea, ether (e.g., —O— between to carbon containinggroups), thioether, sulfinyl, sulfoxide, sulfonyl, thiourea,thiocarbonate, thiocarbamate, ethylene glycol, heterocyclic, triazolefunctionalities or carbamate functionalities such as disclosed in U.S.Pat. No. 5,374,755, and chiral moieties.

The language “chromatographically-enhancing pore geometry” includes thegeometry of the pore configuration of the presently-disclosed materials,which has been found to enhance the chromatographic separation abilityof the material, e.g., as distinguished from other chromatographic mediain the art. For example, a geometry can be formed, selected orconstructed, and various properties and/or factors can be used todetermine whether the chromatographic separations ability of thematerial has been “enhanced,” e.g., as compared to a geometry known orconventionally used in the art. Examples of these factors include highseparation efficiency, longer column life and high mass transferproperties (as evidenced by, e.g., reduced band spreading and good peakshape.) These properties can be measured or observed usingart-recognized techniques. For example, thechromatographically-enhancing pore geometry of the present porousinorganic/organic hybrid materials is distinguished from the prior artmaterials by the absence of “ink bottle” or “shell shaped” pore geometryor morphology, both of which are undesirable because they, e.g., reducemass transfer rates, leading to lower efficiencies.

Chromatographically-enhancing pore geometry is found in hybrid materialscontaining only a small population of micropores. A small population ofmicropores is achieved in hybrid materials when all pores of a diameterof about <34 Å contribute less than about 110 m²/g to the specificsurface area of the material. Hybrid materials with such a low microporesurface area (MSA) give chromatographic enhancements including highseparation efficiency and good mass transfer properties (as evidencedby, e.g., reduced band spreading and good peak shape). Micropore surfacearea (MSA) is defined as the surface area in pores with diameters lessthan or equal to 34 Å, determined by multipoint nitrogen sorptionanalysis from the adsorption leg of the isotherm using the BJH method.As used herein, the acronyms “MSA” and “MPA” are used interchangeably todenote “micropore surface area.”

The term “functionalizing group” includes organic functional groupswhich impart a certain chromatographic functionality to achromatographic stationary phase.

The term “heterocyclic group” includes closed ring structures in whichone or more of the atoms in the ring is an element other than carbon,for example, nitrogen, sulfur, or oxygen. Heterocyclic groups can besaturated or unsaturated and heterocyclic groups such as pyrrole andfuran can have aromatic character, i.e., “heterocyclic aromatic group”.They include one or more ring structures. Heterocyclic groups having twoor more ring structures are “polyheterocyclic aromatic groups”. Thesegroups can have fused ring structures such as quinoline andisoquinoline. Other examples of heterocyclic groups include pyridine andpurine. Heterocyclic groups can also be substituted at one or moreconstituent atoms with, for example, a halogen, a lower alkyl, a loweralkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a loweralkylcarboxyl, a nitro, a hydroxyl, —CF₃, —CN, or the like. Suitableheteroaromatic and heteroalicyclic groups generally will have 1 to 3separate or fused rings with 3 to about 8 members per ring and one ormore N, O or S atoms, e.g., coumarinyl, quinolinyl, pyridyl, pyrazinyl,pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl,indolyl, benzofuranyl, benzothiazolyl, tetrahydrofuranyl,tetrahydropyranyl, piperidinyl, morpholino and pyrrolidinyl.

The term “metal oxide precursor” is intended to include any compoundthat contains a metal and results in the formation of a metal oxide,e.g., alumina, silica, titanium oxide, zirconium oxide.

The term “monolith” is intended to include a collection of individualparticles packed into a bed formation, in which the shape and morphologyof the individual particles are maintained. The particles areadvantageously packed using a material that binds the particlestogether. Any number of binding materials that are well known in the artcan be used such as, for example, linear or cross-linked polymers ofdivinylbenzene, methacrylate, urethanes, alkenes, alkynes, amines,amides, isocyanates, or epoxy groups, as well as condensation reactionsof organoalkoxysilanes, tetraalkoxysilanes, polyorganoalkoxysiloxanes,polyethoxysiloxanes, and ceramic precursors. In certain embodiments, theterm “monolith” also includes hybrid monoliths made by other methods,such as hybrid monoliths detailed in U.S. Pat. No. 7,250,214; hybridmonoliths prepared from the condensation of one or more monomers thatcontain 0-99 mole percent silica (e.g., SiO₂); hybrid monoliths preparedfrom coalesced porous inorganic/organic particles; hybrid monoliths thathave a chromatographically-enhancing pore geometry; hybrid monolithsthat do not have a chromatographically-enhancing pore geometry; hybridmonoliths that have ordered pore structure; hybrid monoliths that havenon-periodic pore structure; hybrid monoliths that have non-crystallineor amorphous molecular ordering; hybrid monoliths that have crystallinedomains or regions; hybrid monoliths with a variety of differentmacropore and mesopore properties; and hybrid monoliths in a variety ofdifferent aspect ratios. In certain embodiments, the term “monolith”also includes inorganic monoliths, such as those described in G.Guiochon/J. Chromatogr. A 1168 (2007) 101-168.

The term “nanoparticle” is a microscopic particle/grain or microscopicmember of a powder/nanopowder with at least one dimension less thanabout 100 nm, e.g., a diameter or particle thickness of less than about100 nm (0.1 mm), which can be crystalline or noncrystalline.Nanoparticles have properties different from, and often superior to,those of conventional bulk materials including, for example, greaterstrength, hardness, ductility, sinterability, and greater reactivityamong others. Considerable scientific study continues to be devoted todetermining the properties of nanomaterials, small amounts of which havebeen synthesized (mainly as nano-size powders) by a number of processesincluding colloidal precipitation, mechanical grinding, and gas-phasenucleation and growth. Extensive reviews have documented recentdevelopments in nano-phase materials, and are incorporated herein byreference thereto: Gleiter, H. (1989) “Nano-crystalline materials,”Prog. Mater. Sci. 33:223-315 and Siegel, R. W. (1993) “Synthesis andproperties of nano-phase materials,” Mater. Sci. Eng. A168:189-197. Incertain embodiments, the nanoparticles comprise oxides or nitrides ofthe following: silicon carbide, aluminum, diamond, cerium, carbon black,carbon nanotubes, zirconium, barium, cerium, cobalt, copper, europium,gadolinium, iron, nickel, samarium, silicon, silver, titanium, zinc,boron, and mixtures thereof. In certain embodiments, the nanoparticlesof the present disclosure are selected from diamonds, zirconium oxide(amorphous, monoclinic, tetragonal and cubic forms), titanium oxide(amorphous, anatase, brookite and rutile forms), aluminum (amorphous,alpha, and gamma forms), and boronitride (cubic form). In particularembodiments, the nanoparticles of the present disclosure are selectedfrom nano-diamonds, silicon carbide, titanium dioxide (anatase form),cubic-boronitride, and any combination thereof. Moreover, in particularembodiments, the nanoparticles can be crystalline or amorphous. Inparticular embodiments, the nanoparticles are less than or equal to 100mm in diameter, e.g., less than or equal to 50 mm in diameter, e.g.,less than or equal to 20 mm in diameter.

Moreover, it should be understood that the nanoparticles that arecharacterized as dispersed within the composites of the presentdisclosure are intended to describe exogenously added nanoparticles.This is in contrast to nanoparticles, or formations containingsignificant similarity with putative nanoparticles, that are capable offormation in situ, wherein, for example, macromolecular structures, suchas particles, can comprise an aggregation of these endogenously created.

The term “substantially disordered” refers to a lack of pore orderingbased on x-ray powder diffraction analysis. Specifically, “substantiallydisordered” is defined by the lack of a peak at a diffraction angle thatcorresponds to a d value (or d-spacing) of at least 1 nm in an x-raydiffraction pattern.

“Surface modifiers” include, typically, organic functional groups whichimpart a certain chromatographic functionality to a chromatographicstationary phase. The porous inorganic/organic hybrid materials possessboth organic groups and silanol groups which can additionally besubstituted or derivatized with a surface modifier.

The language “surface modified” is used herein to describe the compositematerial of the present disclosure that possess both organic groups andsilanol groups which can additionally be substituted or derivatized witha surface modifier. “Surface modifiers” include (typically) organicfunctional groups which impart a certain chromatographic functionalityto a chromatographic stationary phase. Surface modifiers such asdisclosed herein are attached to the base material, e.g., viaderivatization or coating and later crosslinking, imparting the chemicalcharacter of the surface modifier to the base material. In oneembodiment, the organic groups of a hybrid material react to form anorganic covalent bond with a surface modifier. The modifiers can form anorganic covalent bond to the material's organic group via a number ofmechanisms well known in organic and polymer chemistry including but notlimited to nucleophilic, electrophilic, cycloaddition, free-radical,carbene, nitrene, and carbocation reactions. Organic covalent bonds aredefined to involve the formation of a covalent bond between the commonelements of organic chemistry including but not limited to hydrogen,boron, carbon, nitrogen, oxygen, silicon, phosphorus, sulfur, and thehalogens. In addition, carbon-silicon and carbon-oxygen-silicon bondsare defined as organic covalent bonds, whereas silicon-oxygen-siliconbonds that are not defined as organic covalent bonds. A variety ofsynthetic transformations are well known in the literature, see, e.g.,March, J. Advanced Organic Chemistry, 3rd Edition, Wiley, New York,1985.

Chromatographic materials of the present disclosure can include thosecomprising a silica core material, metal oxide core material, aninorganic-organic hybrid material or a group of block copolymers thereofcore material. The core material can be a high purity chromatographiccore composition as discussed herein. Similarly, the chromatographiccore material can be a regular, e.g., not high purity,version/analog/homolog of the high purity materials discussed herein.

Examples of suitable core materials include, but are not limited to,conventional chromatographic silica materials, metal oxide materials,inorganic-organic hybrid materials or a group of block copolymersthereof, ceramic, silicon oxide, silicon imidonitride, silicon nitride,silicon aluminum nitride, silicon diimide, and silicon oxynitride.Additional examples of suitable core materials (for use with or withoutmodification) are described in US Pub. Nos. 2009/0127177, 2007/0135304,2009/0209722, 2007/0215547, 2007/0141325, 2011/0049056, 2012/0055860,and 2012/0273404 as well as International Pub. No. WO2008/103423, whichare incorporated herein by reference in their entirety.

The chromatographic core material can be in the form of discreetparticles or can be a monolith. The chromatographic core material can beany porous material and can be commercially available or can be producedby known methods, such as those methods described in, for example, inU.S. Pat. Nos. 4,017,528, 6,528,167, 6,686,035 and 7,175,913, which areincorporated herein by reference in their entirety. In some embodiments,the chromatographic core material can be a non-porous core.

The composition of the chromatographic surface material and thechromatographic core material can be varied by one of ordinary skill inthe art to provide enhanced chromatographic selectivity, enhanced columnchemical stability, enhanced column efficiency, and/or enhancedmechanical strength. Similarly, the composition of the surroundingmaterial provides a change in hydrophilic/lipophilic balance (HLB),surface charge (e.g., isoelectric point or silanol pK_(a)), and/orsurface functionality for enhanced chromatographic separation.Furthermore, in some embodiments, the composition of the chromatographicmaterial can also provide a surface functionality for available forfurther surface modification.

The ionizable groups and the hydrophobic surface groups of thechromatographic materials of the present disclosure can be preparedusing known methods. Some of the ionizable modifier reagents arecommercially available. For example, silanes having amino alkyltrialkoxysilanes, methyl amino alkyl trialkoxysilanes, and pyridyl alkyltrialkoxysilanes are commercially available. Other silanes such aschloropropyl alkyl trichlorosilane and chloropropyl alkyltrialkoxysilane are also commercially available. These can be bonded andreacted with imidazole to create imidazolyl alkyl silyl surface species,or bonded and reacted with pyridine to create pyridyl alkyl silylsurface species. Other acidic modifiers are also commercially available,including, but not limited to, sulfopropyltrisilanol,carboxyethylsilanetriol, 2-(carbomethoxy) ethylmethyldichlorosilane,2-(carbomethoxy) ethyltrichlorosilane, 2-(carbomethoxy)ethyltrimethoxysilane, n-(trimethoxysilylpropyl) ethylenediamine,triacetic acid, (2-diethylphosphatoethyl) triethoxysilane,2-(chlorosulfonylphenyl) ethyltrichlorosilane, and2-(chlorosulfonylphenyl) ethyltrimethoxysilane.

It is known to one skilled in the art to synthesize these types ofsilanes using common synthetic protocols, including Grignard reactionsand hydrosilylations. Products can be purified by chromatography,recrystallization or distillation.

Other additives such as isocyanates are also commercially available orcan be synthesized by one skilled in the art. A common isocyanateforming protocol is the reaction of a primary amine with phosgene or areagent known as triphosgene.

In one aspect, the present disclosure relates to a method of separatinga compound of interest from a mixture, the method comprising (a)providing a mixture containing the compound of interest; (b) introducinga portion of the mixture to a chromatographic system having achromatographic column; and (c) eluting the separated compound ofinterest from the column; wherein the column has a stationary phasehaving the following structure (i):[X](W)_(a)(Q)_(b)(T)_(c)  (i)

wherein:

X is a chromatographic substrate containing silica, metal oxide, aninorganic-organic hybrid material, a group of block copolymers, orcombinations thereof;

W is selected from the group consisting of hydrogen and hydroxyl,wherein W is bound to the surface of X;

Q is a first substituent which minimizes analyte retention variationover time under chromatographic conditions having low waterconcentrations;

T is a second substituent which chromatographically retains the analyte,wherein T has one or more mono-aromatic, polyaromatic, heterocyclicaromatic, or polyheterocyclic aromatic groups, each group beingoptionally substituted with an aliphatic group; and

b and c are positive numbers, 0.05≤(b/c)≤100, and a ≥0.

In various embodiments, the selectivity of a chromatographic materialcan be controlled or influenced through the selection of Q and/or T, thedensity of Q and/or T on the surface, or a combination thereof. In someembodiments, both Q and T play a role in retaining the compound ofinterest. In other embodiments, T selectively retains each differentcompound of interest.

In various embodiments, the present disclosure provides a bondedchromatographic material to which ligands can be attached. The highdensity coverage attained by the bonding method of the present inventioncan be 2× to 3× higher than silane bonding chemistries currentlypracticed in conventional SFC materials. A combination of high coverageand the other properties of Q and/or T can prevent the interaction ofsurface silanols with analytes.

In various embodiments, the present disclosure provides high density ofbonded phases increases retention and prevents retention drift or changecaused by analyte interactions with surface silanols or other secondaryretention mechanisms. Elimination of these secondary and minorselectivity components greatly improves peak shape and column peakcapacity, especially for basic analytes.

In various embodiments, the present disclosure provides for the use of acoupling chemistry based two component system produces an even coverageof mixed surface functionality. Unlike mixed particle beds where twoseparate particles with different surface chemistries are mixed thismaterial has an even, and predictable, surface character throughout. Acolumn packed with such material is not prone to chromatographicinstability due to poor particle mixing or particle type segregationduring column packing.

In various embodiments, the present disclosure provides for columnpacking that is simplified due to the use of a single particle slurry.

In various embodiments, the present disclosure provides for achromatographic packing material with enhanced selectivity for acidic,neutral and basic analytes in a single column without the use of a mixedor multiple particle based bed.

In various embodiments, the present disclosure provides for a ratio ofthe components on the particle surface that can be easily manipulated toalter the selectivity of the support providing a wide range of optionsfor chromatographic separation.

In various embodiments, the present disclosure provides for bondingchemistry whereby the formation of a crosslinked film of the silanesurface modifying agent either through polymerization of the surfacereactive groups or by the addition of a cross linking agent eitherbefore during or after the addition of the selectivity ligand.

In certain other embodiments, the chromatographic material of thepresent disclosure is non-porous. In another embodiment, thechromatographic material of the present disclosure is hydrolyticallystable at a pH of about 1 to about 14; at a pH of about 10 to about 14;or at a pH of about 1 to about 5.

In another aspect, the present disclosure provides materials asdescribed herein wherein the chromatographic material further comprisesa nanoparticle or a mixture of more than one nanoparticles dispersedwithin the chromatographic surface.

In certain embodiments, the nanoparticle is present in <20% by weight ofthe nanocomposite, <10% by weight of the nanocomposite, or <5% by weightof the nanocomposite.

In other embodiments, the nanoparticle is crystalline or amorphous andcan be silicon carbide, aluminum, diamond, cerium, carbon black, carbonnanotubes, zirconium, barium, cerium, cobalt, copper, europium,gadolinium, iron, nickel, samarium, silicon, silver, titanium, zinc,boron, oxides thereof, or a nitride thereof. In particular embodiments,the nanoparticle is a substance which comprises one or more moietiesselected from the group consisting of nano-diamonds, silicon carbide,titanium dioxide, and cubic-boronitride. In other embodiments, thenanoparticles can be less than or equal to 200 nm in diameter, less thanor equal to 100 nm in diameter, less than or equal to 50 nm in diameter,or less than or equal to 20 nm in diameter.

In one or more embodiments, Q is represented by:

wherein:

n¹ is an integer from 1-30;

n² is an integer from 1-30;

R¹, R², R³ and R⁴ are each independently selected from the groupconsisting of hydrogen, hydroxyl, fluoro, methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, tert-butyl, lower alkyl, a protected ordeprotected alcohol, and a zwitterion;

Z is

(a) a surface attachment group having the formula(B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si—, wherein x is an integer from 1-3, y is aninteger from 0-2, z is an integer from 0-2, and x+y+z=3; R⁵ and R⁶ areeach independently selected from the group consisting of methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, substituted orunsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, aprotected or deprotected alcohol, a zwitterion group and a siloxanebond; and B¹ is a siloxane bond;

(b) an attachment to a surface organofunctional hybrid group through adirect carbon-carbon bond formation or through a heteroatom, ester,ether, thioether, amine, amide, imide, urea, carbonate, carbamate,heterocycle, triazole, or urethane linkage; or

(c) an adsorbed, surface group that is not covalently attached to thesurface of the material;

Y is an embedded polar functionality; and

A is selected from the group consisting of an hydrophilic terminalgroup, a functionizable group, hydrogen, hydroxyl, fluoro, methyl,ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, a loweralkyl and a polarizable group.

In one or more embodiments, T is represented by:

wherein

m¹ is an integer from 1-30;

m² is an integer from 1-30;

m³ is an integer from 1-3;

R⁷, R⁸, R⁹ and R¹⁰ are each independently selected from the groupconsisting of hydrogen, hydroxyl, fluoro, methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, tert-butyl, lower alkyl, a protected ordeprotected alcohol, a zwitterion, an aromatic hydrocarbon group and aheterocyclic aromatic hydrocarbon group;

Z is

(a) a surface attachment group having the formula(B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si—, wherein x is an integer from 1-3, y is aninteger from 0-2, z is an integer from 0-2, and x+y+z=3; R⁵ and R⁶ areeach independently selected from the group consisting of methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, substituted orunsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, aprotected or deprotected alcohol, a zwitterion group and a siloxanebond; and B¹ is a siloxane bond;

(b) an attachment to a surface organofunctional hybrid group through adirect carbon-carbon bond formation or through a heteroatom, ester,ether, thioether, amine, amide, imide, urea, carbonate, carbamate,heterocycle, triazole, or urethane linkage; or

(c) an adsorbed, surface group that is not covalently attached to thesurface of the material;

Y is an embedded polar functionality;

D is selected from the group consisting of a bond, N, O, S,

—(CH₂)₀₋₁₂—N—R¹¹R¹²,

—(CH₂)₀₋₁₂—O—R¹¹,

—(CH₂)₀₋₁₂—S—R¹¹,

—(CH₂)₀₋₁₂—N—(CH₂)₀₋₁₂—R¹¹R¹²,

—(CH₂)₀₋₂—O—(CH₂)₀₋₂—R¹¹,

—(CH₂)₀₋₁₂—S—(CH₂)₀₋₁₂—R¹¹,

—(CH₂)₀₋₁₂—S(O)₁₋₂—(CH₂)₀₋₁₂—N—R¹¹R¹²,

—(CH₂)₀₋₁₂—S(O)₁₋₂—(CH₂)₀₋₁₂—O—R¹¹,

—(CH₂)₀₋₁₂—S(O)₁₋₂—(CH₂)₀₋₁₂—S—R¹¹;

—(CH₂)₀₋₁₂—S(O)₁₋₂—(CH₂)₀₋₁₂—N—(CH₂)₀₋₁₂—R¹¹R¹²,

—(CH₂)₀₋₁₂—S(O)₁₋₂—(CH₂)₀₋₁₂—O—(CH₂)₀₋₁₂—R¹¹, and

—(CH₂)₀₋₁₂—S(O)₁₋₂—(CH₂)₀₋₁₂—S—(CH₂)₀₋₁₂—R¹¹,

R¹¹ is a first mono-aromatic, polyaromatic, heterocyclic aromatic, orpolyheterocyclic aromatic group; and

R¹² is a hydrogen, an aliphatic group or a second mono-aromatic,polyaromatic, heterocyclic aromatic, or polyheterocyclic aromatic group,wherein R¹¹ and R¹² are optionally substituted with one or more groupsselected from the group consisting of an aliphatic group, a halogen, ahydroxyl, a thiol, an amino, an alkoxy, an alkylcarboxy, an alkylthio,and a nitro group.

In some embodiments, the first or second mono-aromatic, polyaromatic,heterocyclic aromatic, or polyheterocyclic aromatic group of R¹¹ or R¹²can be a polyaromatic or polyhetercyclic aromatic hydrocarbon with atleast 2 aromatic rings. The first or second mono-aromatic, polyaromatic,heterocyclic aromatic, or polyheterocyclic aromatic group of R¹¹ or R¹²can also be a polyaromatic or polyhetercyclic aromatic hydrocarbon withat least 3 aromatic rings. The first or second mono-aromatic,polyaromatic, heterocyclic aromatic, or polyheterocyclic aromatic groupof R¹¹ or R¹² can also be a polyaromatic or polyhetercyclic aromatichydrocarbon with at least 4 aromatic rings.

In one embodiment, the first or second mono-aromatic, polyaromatic,heterocyclic aromatic, or polyheterocyclic aromatic group of R¹¹ or R¹²is selected from the group consisting of furan, pyrrole, pyrroline,oxazole, thiazole, imidazole, imidazoline, pyrazole, pyrazoline,pyrazolidine, isoxazole, isothiazole, benzene, pyridine, pyridazine,pyrimidine, pyrazine, triazine, thiophene, indene, indolinzine, indole,isoindole, indoline, indazole, benzimidazole, benzthiazole, naphthalene,quinolizine, quinoline, isoquinoline, cinnoline, phthalazine,quinazoline, quinoxaline, 1,8-naphthyridine, quinuclidine, fluorene,carbazole, anthracene, acridine, phanazine, phenothiazine, phenoxazine,pyrene, and derivatives thereof, wherein the group is unsubstituted oroptionally substituted with a aliphatic group.

In other embodiments, the first or second mono-aromatic, polyaromatic,heterocyclic aromatic, or polyheterocyclic aromatic group of R¹¹ or R¹²can be substituted with at least one C₁-C₂₄ aliphatic group. Inparticular, the groups can be substituted with at least one C₂-C₂₂aliphatic group, one C₃-C₂₀ aliphatic group, one C₄-C₁₈ aliphatic group,one C₅-C₁₆ aliphatic group, one C₆-C₁₄ aliphatic group, one C₇-C₁₂aliphatic group, one C₈-C₁₀ aliphatic group, or one aliphatic group ofany combination of preceding carbon lengths, e.g. a C₈-C₁₈ aliphaticgroup, or other various sized groups as described in the presentdisclosure.

R11 or R12 can be an aminoanthracene (e.g., 1-aminoanthracene,2-aminoanthracene or 9-aminoanthracene) or a methylaminoanthracene (e.g.1-methylaminoanthracene, 2-methylaminoanthracene or9-methylaminoanthracene). The aminoanthracene or methylaminoanthracenecan be substituted on the ring structure with one more aliphatic groups,for example, a lower alkyl. In some embodiments, the aminoanthracene ormethylaminoanthracene can have the formula(X)-amino-(Y)-alkyl-anthracene or (X)-methylamino-(Y)-alkyl-anthracenewherein X is 1, 2 or 9 and Y is 1-10 representing the carbon positionson anthracene (e.g., 1-amino-1-methyl-anthracene;1-amino-2-methyl-anthracene; 1-amino-3-methyl-anthracene;1-amino-4-methyl-anthracene; 1-amino-5-methyl-anthracene;1-amino-6-methyl-anthracene; 1-amino-7-methyl-anthracene;1-amino-8-methyl-anthracene; 1-amino-9-methyl-anthracene;1-amino-10-methyl-anthracene; 1-methylamino-1-methyl-anthracene;1-methylamino-2-methyl-anthracene; 1-methylamino-3-methyl-anthracene;1-methylamino-4-methyl-anthracene; 1-methylamino-5-methyl-anthracene;1-methylamino-6-methyl-anthracene; 1-methylamino-7-methyl-anthracene;1-methylamino-8-methyl-anthracene; 1-methylamino-9-methyl-anthracene;and 1-methylamino-10-methyl-anthracene, etc.).

In other embodiments, the aminoanthracene or methylaminoanthracene canhave the formula (X)-amino-(Y)-alkyl-(Z)-alkyl-anthracene or(X)-methylamino-(Y)-alkyl-(Z)-alkyl-anthracene wherein X is 1, 2 or 9and, Y and Z are 1-10 representing the carbon positions on anthracene,provided Y and Z are not the same (e.g.,1-amino-1-methyl-2-methyl-anthracene;1-amino-1-methyl-3-methyl-anthracene;1-amino-1-methyl-4-methyl-anthracene;1-amino-1-methyl-5-methyl-anthracene;1-amino-1-methyl-6-methyl-anthracene;1-amino-1-methyl-7-methyl-anthracene;1-amino-1-methyl-8-methyl-anthracene;1-amino-1-methyl-9-methyl-anthracene;1-amino-1-methyl-10-methyl-anthracene; etc.). The aminoanthracene ormethylaminoanthracene can be disubstituted on the ring structure with asecond polar group, for example, an amine (e.g., 1-amino,4-N,N-dimethylaminoanthracene).

R11 or R12 can be a naphthylamine (e.g., 1-naphthylamine, or2-naphthylamine) or a methylnaphthylamine (e.g., 1-methylnaphthylamine,or 2-methylnaphthylamine). The naphthylamine or methylnaphthylamine canbe substituted with one more aliphatic groups, for example, a loweralkyl. In some embodiments, the naphthylamine or methylnaphthylamine canhave the formula (X′)-amino-(Y′)-alkyl-naphthalene or(X′)-methylamino-(Y′)-alkyl-naphthalene wherein X′ is 1 or 2 and Y′ is1-8 representing the carbon positions on naphthalene (e.g.,1-amino-1-methyl-naphthalene; 1-amino-2-methyl-naphthalene;1-amino-3-methyl-naphthalene; 1-amino-4-methyl-naphthalene;1-amino-5-methyl-naphthalene; 1-amino-6-methyl-naphthalene;1-amino-7-methyl-naphthalene; 1-amino-8-methyl-naphthalene; 9 and 10positions are non-substitutable; 1-methylamino-1-methyl-naphthalene;1-methylamino-2-methyl-naphthalene; 1-methylamino-3-methyl-naphthalene;1-methylamino-4-methyl-naphthalene; 1-methylamino-5-methyl-naphthalene;1-methylamino-6-methyl-naphthalene; 1-methylamino-7-methyl-naphthalene;1-methylamino-8-methyl-naphthalene; etc.).

In other embodiments, the naphthylamine or methylnaphthylamine can havethe formula (X′)-amino-(Y′)-alkyl-(Z′)-alkyl-naphthalene or(X′)-methylamino-(Y′)-alkyl-(Z′)-alkyl-naphthalene wherein X′ is 1 or 2and, Y′ and Z′ are 1-10 representing the carbon positions onnaphthalene, provided Y′ and Z′ are not the same (e.g.,1-amino-1-methyl-2-methyl-naphthalene;1-amino-1-methyl-3-methyl-naphthalene;1-amino-1-methyl-4-methyl-naphthalene;1-amino-1-methyl-5-methyl-naphthalene;1-amino-1-methyl-6-methyl-naphthalene;1-amino-1-methyl-7-methyl-naphthalene;1-amino-1-methyl-8-methyl-naphthalene; etc.).

R11 or R12 can be an aminophenanthrene (e.g., 1-aminophenanthrene,2-aminophenanthrene, 3-aminophenanthrene, 4-aminophenanthrene or9-aminophenanthrene) or a methylaminophenanthrene (e.g.,1-methylaminophenanthrene, 2-methylaminophenanthrene,3-methylaminophenanthrene, 4-methylaminophenanthrene or9-methylaminophenanthrene). The aminophenanthrene ormethylaminophenanthrene can be substituted on the ring structure withone more aliphatic groups, for example, a lower alkyl. In someembodiments, the aminophenanthrene or methylaminophenanthrene can havethe formula (X″)-amino-(Y″)-alkyl-phenanthrene or(X″)-methylamino-(Y″)-alkyl-phenanthrene wherein X is 1, 2, 3, 4 or 9and Y is 1-10 representing the carbon positions on phenanthrene (e.g.,1-amino-1-methyl-phenanthrene; 1-amino-2-methyl-phenanthrene;1-methylamino-1-methyl-phenanthrene;1-methylamino-2-methyl-phenanthrene; etc.).

In other embodiments, the aminophenanthrene or methylaminophenanthrenecan have the formula (X″)-amino-(Y″)-alkyl-(Z″)-alkyl-phenanthrene or(X″)-methylamino-(Y″)-alkyl-(Z″)-alkyl-phenanthrene wherein X is 1, 2,3, 4 or 9 and, Y and Z are 1-10 (not counting the junction carbons)representing the carbon positions on phenanthrene, provided Y and Z arenot the same (e.g., 1-amino-1-methyl-2-methyl-phenanthrene; etc.).

Similarly, R11 or R12 can be an aminopyrene (e.g., 1-aminopyrene,2-aminopyrene, 3-aminopyrene, 4-aminopyrene or 5-aminopyrene) or amethylaminopyrene (e.g., 1-methylaminopyrene, 2-methylaminopyrene,3-methylaminopyrene, 4-methylaminopyrene or 5-methylaminopyrene). Theaminopyrene and methylaminopyrene can be similarly substituted as theaminoanthracene and methylaminoanthracene.

Similarly, R11 or R12 can be an aminochrysene (e.g., 1-aminochrysene,2-aminochrysene, 3-aminochrysene, 4-aminochrysene, 5-aminochrysene, or6-aminochrysene) or a methylaminochrysene (e.g., 1-methylaminochrysene,2-methylaminochrysene, 3-methylaminochrysene, 4-methylaminochrysene,5-methylaminochrysene, or 6-methylaminochrysene). The aminochrysene andmethylaminochrysene can be similarly substituted as the aminoanthraceneand methylaminoanthracene.

In some embodiments, T is represented by one of the followingstructures:

wherein each E is independently a bond or a lower alkyl that can beoptionally substituted with an hydroxyl, fluoro, methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, lower alkyl, aprotected or deprotected alcohol, a zwitterion, an aromatic hydrocarbongroup or a heterocyclic aromatic hydrocarbon group, and wherein Z, Y,R⁹, m¹, m² and D are defined above.

In other embodiments, T is represented by one of the followingstructures:

wherein Z, Y, R⁹, m¹, m² and D are defined above.

In some embodiments, b and c are positive numbers, with a ratio0.05≤(b/c)≤100, and a ≥0. In some embodiments, Q and T are different,whereas in other embodiments Q and T are the same. Q can include two ormore different moieties, and T can include two or more differentmoieties. In some embodiments, the first, second, third, fourth, andfifth fraction are each independently about 0-100, 1-99, 5-95, 10-90,20-80, 30-70, 40-60, 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, or 95%.

In one or more embodiments, Q is non-polar. In some embodiments, Qcomprises a borate or nitro functional group. In some embodiments, Q isrepresented by one of:

wherein Z can include a surface attachment group having the formula(B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si—, wherein x is an integer from 1-3, y is aninteger from 0-2, z is an integer from 0-2, and x+y+z=3. Each occurrenceof R⁵ and R⁶ can independently represent methyl, ethyl, n-butyl,iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstitutedaryl, cyclic alkyl, branched alkyl, lower alkyl, a protected ordeprotected alcohol, or a zwitterion group, and B¹ can represent asiloxane bond.

In another embodiment, Z is an attachment to a surface organofunctionalhybrid group through a direct carbon-carbon bond formation or through aheteroatom, ester, ether, thioether, amine, amide, imide, urea,carbonate, carbamate, heterocycle, triazole, or urethane linkage. In yetanother embodiment, Z is an adsorbed, surface group that is notcovalently attached to the surface of the material.

In some embodiments, T is represented by one of:

wherein Z can include a surface attachment group having the formula(B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si—, wherein x is an integer from 1-3, y is aninteger from 0-2, z is an integer from 0-2, and x+y+z=3. Each occurrenceof R⁵ and R⁶ can independently represent methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, tert-butyl, substituted or unsubstitutedaryl, cyclic alkyl, branched alkyl, lower alkyl, a protected ordeprotected alcohol, a zwitterion group and a siloxane bond, and B¹ canrepresent a siloxane bond. In some embodiments, Z is an attachment to asurface organofunctional hybrid group through a direct carbon-carbonbond formation or through a heteroatom, ester, ether, thioether, amine,amide, imide, urea, carbonate, carbamate, heterocycle, triazole, orurethane linkage. In some embodiments, Z is an adsorbed, surface groupthat is not covalently attached to the surface of the material.

In some embodiments, the first or second mono-aromatic, polyaromatic,heterocyclic aromatic, or polyheterocyclic aromatic group of R¹¹ or R¹²can be converted to a cycloolefin. For example, a pyridine group may beconverted to a cycloolefin in solution under certain conditions. In somecircumstances, the cycloolefin retains sufficient unsaturation to retainand separate structurally related compound(s) of interest from amixture. In particular situations, a stationary phase of the presentdisclosure containing the cycloolefin can retain, separate andresolution of critical pairs related to vitamins (e.g., D2 and D3, K1and K2).

The Q and T substituents can also be polymerized. The Q and Tsubstituents can be polymerized to each itself, e.g., Q-Q, T-T, orpolymerized to each other, e.g. Q-T. As illustrated in FIG. 4,polymerization can occur at the surface level between the siloxanegroups, between the hydrocarbon substituents, or both. Polymerizationbetween the substituents can create a crosslinked surface coating, or asecond coating above the surface coating, e.g. a second layer ofsubstituents which may or may not also be polymerized, or a mixture ofboth. The degree of polymerization of the Q and T substituents can vary.For example, a first fraction of Q can be bound to X and a secondfraction of Q can be polymerized. Likewise, a first fraction of T can bebound to X, and a second fraction of T can be polymerized. In anotherembodiment, a first fraction of Q can be bound to X, a second fractionof Q can be polymerized, a third fraction of T can be bound to X, and afourth fraction of T can be polymerized. The polymerized portions of Qand T can be polymerized to itself or to each other.

In one or more embodiments of any of the above aspects, X is a highpurity chromatographic material having a core surface that is subject toalkoxylation by a chromatographic mobile phase under chromatographicconditions. X can be a chromatographic material having a core surfacethat is subject to alkoxylation by a chromatographic mobile phase underchromatographic conditions. In some embodiments, the functional groupincluding Q is a diol. The functional group including T can be an amine,an ether, a thioether, or a combination thereof. T can include a chiralfunctional group adapted for a chiral separation, Q can include a chiralfunctional group adapted for a chiral separation, or T and Q can bothinclude a chiral functional group adapted for a chiral separation.

In one or more embodiments of the above aspects, the ratio b/c is about0.05-75, 0.05-50, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90. In some embodiments,the surface of X does not include silica, and b=0 or c=0. In someembodiments, the combined surface coverage is greater than about 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4, 5, 6, 7, or 8 μmol/m².

In some embodiments of the above aspects, the chromatographic stationaryphase exhibits a retention drift or change of ≤5% over 30 days, ≤4% over30 days, ≤3% over 30 days, ≤2% over 30 days, ≤1% over 30 days, ≤5% over10 days, ≤4% over 10 days, ≤3% over 10 days, ≤2% over 10 days, ≤1% over10 days, ≤5% over 3 days, ≤4% over 3 days, ≤3% over 3 days, ≤2% over 3days, ≤1% over 3 days, ≤5% over 30 runs, ≤4% over 30 runs, ≤3% over 30runs, ≤2% over 30 runs, ≤1% over 30 runs, ≤5% over 10 runs, ≤4% over 10runs, ≤3% over 10 runs, ≤2% over 10 runs, ≤1% over 10 runs, ≤5% over 3runs, ≤4% over 3 runs, ≤3% over 3 runs, ≤2% over 3 runs, or ≤1% over 3runs.

In some embodiments, the core material consists essentially of a silicamaterial. Optionally, the core material consists essentially of anorganic-inorganic hybrid material or a superficially porous material. Inone or more embodiments, the core material consists essentially of aninorganic material with a hybrid surface layer, a hybrid material withan inorganic surface layer, a surrounded hybrid layer, or a hybridmaterial with a different hybrid surface layer. The stationary phasematerial can optionally be in the form of a plurality of particles, amonolith, or a superficially porous material. In some embodiments thestationary phase material does not have chromatographically enhancingpore geometry whereas in other embodiments the stationary phase materialhas chromatographically enhancing pore geometry. The stationary phasematerial can be in the form of a spherical material, non-sphericalmaterial (e.g., including toroids, polyhedrons). In certain embodiments,the stationary phase material has a highly spherical core morphology, arod shaped core morphology, a bent-rod shaped core morphology, a toroidshaped core morphology; or a dumbbell shaped core morphology. In certainembodiments, the stationary phase material has a mixture of highlyspherical, rod shaped, bent rod shaped, toroid shaped, or dumbbellshaped morphologies.

In some embodiments, the stationary phase material has a surface area ofabout 25 to 1100 m²/g, about 150 to 750 m²/g, or about 300 to 500 m²/g.In some embodiments, the stationary phase material has a pore volume ofabout 0.2 to 2.0 cm³/g, or about 0.7 to 1.5 cm³/g. In some embodiments,the stationary phase material has a micropore surface area of less thanabout 105 m²/g, less than about 80 m²/g, or less than about 50 m²/g. Thestationary phase material can have an average pore diameter of about 20to 1500 Å, about 50 to 1000 Å, about 60 to 750 Å, or about 65 to 200 Å.In some embodiments, the plurality of particles have sizes between about0.2 and 100 microns, between about 0.5 and 10 microns, or between about1.5 and 5 microns.

In one or more embodiments, X includes a silica core, c=0, and Q has acombined surface coverage of ≥2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0,4.5, or 5 μmol/m²; or X includes a non-silica core or a silica-organichybrid core, c=0, and Q has a combined surface coverage of ≥0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,2.5, 3.0, 3.5, 4.0, 4.5, or 5 μmol/m²; or b≥0, c≥0, and Q has a combinedsurface coverage of ≥0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5 μmol/m².

In other embodiments, Q has a combined surface coverage of ≥0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0μmol/m². In other embodiments, T has a combined surface coverage of≥0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.5, 3.0 or 3.5 μmol/m². T can also have a combined surfacecoverage between about 0.1 and about 4.0 μmol/m², or about 0.2 and about3.9 μmol/m², or about 0.3 and about 3.8 μmol/m², or about 0.4 and about3.7 μmol/m², or about 0.5 and about 3.6 μmol/m², or about 1.0 and about3.5 μmol/m², or about 1.2 and about 3.0 μmol/m², or any combination ofvalues, such as between about 3.0 and about 4.0 μmol/m².

In other embodiments, the overall combined coverage of Q and T is ≥0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.5, 6.0 or 6.5 μmol/m².

The chromatographic stationary phase can be adapted for normal phasechromatography, high-pressure liquid chromatography, solvated gaschromatography, supercritical fluid chromatography, sub-critical fluidchromatography, carbon dioxide based chromatography, hydrophilicinteraction liquid chromatography or hydrophobic interaction liquidchromatography.

The chromatographic stationary phase can include radially adjustedpores, non-radially adjusted pores, ordered pores, non-ordered pores,monodispersed pores, non-monodispersed pores, smooth surfaces, roughsurfaces or combinations thereof. In one or more embodiments, T has oneionizable group, T has more than one ionizable group, T has two or moreionizable groups of the same pKa, or T has two or more ionizable groupof different pKa.

In another embodiment, the present disclosure relates to achromatographic stationary phase having the following structure (i):[X](W)_(a)(Q)_(b)(T)_(c)  (i)

wherein X is a chromatographic substrate containing silica, metal oxide,an inorganic-organic hybrid material, a group of block copolymers, orcombinations thereof; W is selected from the group consisting ofhydrogen and hydroxyl, wherein W is bound to the surface of X; Q is afirst substituent which minimizes analyte retention variation over timeunder chromatographic conditions having low water concentrations; T is asecond substituent which chromatographically retains the analyte,wherein T has one or more aromatic, polyaromatic, heterocyclic aromatic,or polyheterocyclic aromatic hydrocarbon groups, each group beingoptionally substituted with an aliphatic group; and b and c are positivenumbers, 0.05≤(b/c)≤100, and a ≥0.

In another embodiment, the present disclosure relates to a column,capillary column, microfluidic device or apparatus for normal phasechromatography, high-pressure liquid chromatography, solvated gaschromatography, supercritical fluid chromatography, sub-critical fluidchromatography, carbon dioxide based chromatography, hydrophilicinteraction liquid chromatography or hydrophobic interaction liquidchromatography comprising a housing having at least one wall defining achamber having an entrance and an exit, and a stationary phase asdescribed in the present disclosure disposed therein, wherein thehousing and stationary phase are adapted for normal phasechromatography, high-pressure liquid chromatography, solvated gaschromatography, supercritical fluid chromatography, sub-critical fluidchromatography, carbon dioxide based chromatography, hydrophilicinteraction liquid chromatography or hydrophobic interaction liquidchromatography.

In another embodiment, the present disclosure relates to a kit fornormal phase chromatography, high-pressure liquid chromatography,solvated gas chromatography, supercritical fluid chromatography,sub-critical fluid chromatography, carbon dioxide based chromatography,hydrophilic interaction liquid chromatography or hydrophobic interactionliquid chromatography comprising a housing having at least one walldefining a chamber having an entrance and an exit, and a stationaryphase as described in the present disclosure disposed therein, whereinthe housing and stationary phase are adapted for normal phasechromatography, high-pressure liquid chromatography, solvated gaschromatography, supercritical fluid chromatography, sub-critical fluidchromatography, carbon dioxide based chromatography, hydrophilicinteraction liquid chromatography or hydrophobic interaction liquidchromatography; and instructions for performing normal phasechromatography, high-pressure liquid chromatography, solvated gaschromatography, supercritical fluid chromatography, sub-critical fluidchromatography, carbon dioxide based chromatography, hydrophilicinteraction liquid chromatography or hydrophobic interaction liquidchromatography with the housing and stationary phase.

The kit can include a housing having at least one wall defining achamber having an entrance and an exit, and a stationary phase accordingto any embodiments of the present disclosure disposed therein. Thedevices can have preformed frits, frits generated by interconnectedmaterials, or devices without frits. The housing and stationary phasecan be adapted for normal phase chromatography, high-pressure liquidchromatography, solvated gas chromatography, supercritical fluidchromatography, sub-critical fluid chromatography, carbon dioxide basedchromatography, hydrophilic interaction liquid chromatography orhydrophobic interaction liquid chromatography, or a combination thereof.Additionally, instructions for performing normal phase chromatography,high-pressure liquid chromatography, solvated gas chromatography,supercritical fluid chromatography, sub-critical fluid chromatography,carbon dioxide based chromatography, hydrophilic interaction liquidchromatography or hydrophobic interaction liquid chromatography, or acombination thereof with the housing and stationary phase can beincluded.

Accordingly, the kit of the present disclosure can be used to implementthe methods of the invention described herein. Additionally, the kits ofthe present invention can be used to analyze a variety of differentsamples and sample types, including those described herein below.

In one or more embodiments, the present invention can contemplate kitscontaining aspects of the present disclosure to reduce or mitigate theeffects of retention drift or change. For instance, a kit can contain achromatography column packed with a stationary phase media of thepresent disclosure. In some embodiments the packed column can be useddirectly in a standard chromatography system (e.g., a commerciallyavailable chromatography system such as a Waters Acquity® chromatographysystem). A kit can further contain instruction for use. Additionally, akit can further contain stock samples of pure analyte for calibration ofthe instrument and/or confirmation of a substantial lack of retentiondrift or change. A kit can include any or all of the componentsdescribed above (e.g., a stationary phase, a packed column, or achromatography apparatus) to mitigate the effects of retention drift orchange.

In another embodiment, the present disclosure relates to a method formitigating or preventing retention drift in normal phase chromatography,high-pressure liquid chromatography, solvated gas chromatography,supercritical fluid chromatography, sub-critical fluid chromatography,carbon dioxide based chromatography, hydrophilic interaction liquidchromatography or hydrophobic interaction liquid chromatographycomprising chromatographically separating a sample using achromatographic device comprising a chromatographic stationary phase asdescribed in the present disclosure, thereby mitigating or preventingretention drift.

In one or more embodiments, mitigating or preventing retention drift orchange includes a retention drift or change of ≤5% over 30 days, ≤4%over 30 days, ≤3% over 30 days, ≤2% over 30 days, ≤1% over 30 days, ≤5%over 10 days, ≤4% over 10 days, ≤3% over 10 days, ≤2% over 10 days, ≤1%over 10 days, ≤5% over 3 days, ≤4% over 3 days, ≤3% over 3 days, ≤2%over 3 days, ≤1% over 3 days, ≤5% over 30 runs, ≤4% over 30 runs, ≤3%over 30 runs, ≤2% over 30 runs, ≤1% over 30 runs, ≤5% over 10 runs, ≤4%over 10 runs, ≤3% over 10 runs, ≤2% over 10 runs, ≤1% over 10 runs, ≤5%over 3 runs, ≤4% over 3 runs, ≤3% over 3 runs, ≤2% over 3 runs, or ≤1%over 3 runs. In some embodiments, mitigating or preventing retentiondrift or change includes substantially eliminating the effect ofalkoxylation and/or dealkoxylation of the chromatographic material onretention.

The concept of chemically modifying chromatographic core materials, asused herein, is understood to include functionalizing a chromatographiccore, for example with a polar silane, or other functional group,thereby mitigating or avoiding retention drift or change. For example,functionalization can essentially prevent chromatographic interactionbetween an analyte and the chromatographic core (e.g., effectivelyeliminating a chromatographic effect of core surface silanols and/oralkoxylated silanols). In some cases, functionalization (e.g., usingnon-polar groups) can reduce the retentivity of the column. Therefore,in various embodiments functionalization of chromatographic corematerials can include the use of hydrophilic, polar, ionizable, and/orcharged functional group that chromatographically interacts with theanalyte, to preserve or achieve a chromatographically useful overallretention. Such endcapping groups can be introduced, for example, viastandard bonding chemistry.

In some embodiments, functionalization provides a permanent attachment.Accordingly, it is important to select an appropriate functionalizationfor the chromatographic phase. In preferred embodiments, thechromatographic material will have chromatographically desirableproperties (e.g., overall retention). Therefore in some embodiments itis important to select a functionalization that has properties that canmimic the desirable (e.g., overall retention) properties of aconventional chromatographic material.

In various embodiments, the chemical properties of a functional groupcan be selected to achieve a desired effect. For example, one or morehydrophilic, polar, ionizable, and/or charged functional group can beused to achieve desired interactions with an analyte (e.g.,chromatographically acceptable retention) and/or the mobile phase (e.g.,repelling alcohols that might alkoxylate a chromatographic coresurface). Likewise, endcap size and/or sterics can be selected to mask acore surface and/or effect a chiral separation.

Similarly, the concentration of functionalization can be varied. In someembodiments, larger and/or more strongly interacting functional groupscan mitigate or avoid retention drift or change at lower concentrations(e.g., as compared to smaller functional groups). In other embodiments,coverage can be tailored for a desired property. For example, nonpolarfunctional groups can be used at lower coverage than polar functionalgroups (e.g., to maintain a desired retention). In various embodiments,functionalization can use one or more polar or nonpolar endcaps, or acombination thereof. In some embodiments, surface area of thechromatographic media is increased or decreased to compensate fordecreased or increased retention due to the altered polarity of thefunctional groups.

In another embodiment, the present disclosure relates to a method forpreparing a stationary phase as described in the present disclosurecomprising reacting a chromatographic substrate with a silane couplingagent having a pendant reactive group; reacting a second chemical agentcomprising one or more aromatic, polyaromatic, heterocyclic aromatic, orpolyheterocyclic aromatic hydrocarbon groups with the pendant reactivegroup; and neutralize any remaining unreacted pendant reactive groups,thereby producing the stationary phase.

In another embodiment, the present disclosure relates to a method forpreparing a stationary phase as described in the present disclosurecomprising oligomerizing a silane coupling agent having a pendantreactive group; reacting a core surface with the oligomerized silanecoupling agent; reacting a second chemical agent comprising one or morearomatic, polyaromatic, heterocyclic aromatic, or polyheterocyclicaromatic hydrocarbon groups with the pendant reactive group; andneutralize any remaining unreacted pendant reactive groups, therebyproducing the stationary phase.

In one or more embodiments, Q is derived from a reagent having one orthe following structures:

In some embodiments, Y includes one of the following structures:

wherein the R⁶ and R⁷ groups associated with the Y group eachindividually is an aliphatic group. In some embodiment, the Y group mayalso be a bond or an aliphatic group.

The above method can be used to make any of the materials (e.g.,chromatography stationary phase materials) as described herein. Forinstance, the methods of the present disclosure can include methods ofreacting a chromatographic stationary phase (e.g., silica particles)with a chemical reagent (e.g., any of the above reagents as describedherein) to chemically modify the surface of the stationary phase tomitigate the effects of retention drift or change.

The present disclosure includes various apparatuses (e.g.,chromatographic columns, capillary and microfluidic devices, and systemsfor use thereof) including the chromatographic materials describedherein. While several illustrative examples are discussed below, apractitioner of ordinary skill will understand that the presentdisclosure can contemplate a number of different embodiments, includingbut not limited to chromatographic columns, apparatuses, methods of use,or kits.

In some embodiments, the present disclosure provides a column orapparatus for normal phase chromatography, high-pressure liquidchromatography, solvated gas chromatography, supercritical fluidchromatography, sub-critical fluid chromatography, carbon dioxide basedchromatography, hydrophilic interaction liquid chromatography orhydrophobic interaction liquid chromatography, or a combination thereof.The column or apparatus includes a housing having at least one walldefining a chamber having an entrance and an exit, as well as astationary phase according to any embodiments of the present disclosuredisposed therein. The devices can have preformed frits, frits generatedby interconnected materials, or devices without frits. The housing andstationary phase can be adapted for normal phase chromatography,high-pressure liquid chromatography, solvated gas chromatography,supercritical fluid chromatography, sub-critical fluid chromatography,carbon dioxide based chromatography, hydrophilic interaction liquidchromatography or hydrophobic interaction liquid chromatography, or acombination thereof.

Accordingly, the apparatus of the present disclosure can contain (e.g.,be packed with) materials of the present disclosure (e.g., achromatographic stationary phase such as a chemically modifiedstationary phase adapted to reduce or mitigate retention drift orchange). Moreover, the apparatus of the present disclosure can be usedto carry out the methods of the present disclosure as described herein.

In one embodiment, the present disclosure is in the form a packedcolumn. The column can be packed with a stationary phase (e.g.,chromatographic material) described herein. Such a column can be used toperform different types of chromatography (e.g., normal phasechromatography, supercritical fluid chromatography, carbon dioxide basedchromatography, hydrophobic interaction liquid chromatography,hydrophilic interaction liquid chromatography, subcritical fluidchromatography, high pressure liquid chromatography, and solvated gaschromatography) while mitigating or avoiding retention drift or change.

The columns can be used in combination with existing chromatographyplatforms such as commercially available chromatography systems,including the Waters Alliance® HPLC system, Waters Acquity® system, orWaters UPC²® system. A column of the present disclosure can be used fora number of different mass throughputs (e.g., analytical scalechromatography, preparative scale chromatography) while mitigating theeffects of retention drift or change. Likewise, the present disclosurecan be embodies in capillary and microfluidic devices, and systems(e.g., commercially available and know to persons of ordinary skill inthe art) for use thereof. The selection of columns, capillary, andmicrofluidic devices, and related systems will be readily understandableto person of ordinary skill in the art.

In various embodiments, material in accordance with the presentdisclosure can have application in microbore columns for use on a SFC,HPLC, and/or UHPLC system. In various embodiments, material inaccordance with the present disclosure can have application fastequilibration columns, long lifetime columns, and SFC with water stablecolumns.

The present disclosure can be used to retain, separate and/or analyze aplurality of different compounds of interest from many different samplesfrom many different fields, for example, from clinical chemistry,medicine, veterinary medicine, forensic chemistry, pharmacology, foodindustry, safety at work, and environmental pollution. The plurality ofsamples including, but are not limited to, small organic molecules,proteins, nucleic acids, lipids, fatty acids, carbohydrates, polymers,and the like. Similarly, the present disclosure can be used for theseparation of small molecules, polar small molecules, analytes used inpharmaceuticals, biomolecules, antibodies, polymers and oligomers,sugars, glycan analysis, petrochemical analysis, lipid analysis,peptides, phosphopeptides, oligonucleotides, DNA, RNA, polar acids,polyaromatic hydrocarbons, food analysis, chemical analysis,bioanalysis, drugs of abuse, forensics, pesticides, agrochemicals,biosimilars, formulations.

Analytes amenable to chromatographic separation with the presentdisclosure can include essentially any molecule of interest, including,for example, small organic molecules, lipids, peptides, nucleic acids,synthetic polymers.

Clinical chemistry target analytes can include any molecule present inan organism (e.g., human body, animal body, fungi, bacterium, virus, andthe like). For example, clinical chemistry target analytes include, butare not limited to, proteins, metabolites, biomarkers, and drugs.

Human medicine and veterinary medicine target analytes can include anymolecule that can be used for the diagnosis, prophylaxis or treatment ofa disease or condition in a subject. For example, human medicine andveterinary medicine target analytes include, but are not limited to,disease markers, prophylactic agents, or therapeutic agents.

Forensic chemistry target analytes can include any molecule present in asample taken from the site of crime, such as a sample from a victim'sbody (e.g., tissue or fluid sample, hair, blood, semen, urine, and thelike). For example, clinical chemistry target analytes include, but arenot limited to, toxic agents, drugs and their metabolites, biomarkers,and identifying compounds.

Pharmacology target analytes can include any molecule that is apharmaceutical or metabolite thereof or which can be used for thedesign, synthesis, and monitoring of drugs. For example, pharmacologytarget analytes include, but are not limited to, prophylactic and/ortherapeutic agents, their prodrugs, intermediates and metabolites.Pharmacological analysis can include bioequivalence testing, forexample, in connection with the approval, manufacturing, and monitoringof a generic drug.

Food industry and agricultural target analytes can include any moleculethat is relevant for monitoring of the safety of foods, beverages,and/or other food industry/agricultural products. Examples of targetanalytes from the field of food industry include, but are not limitedto, pathogen markers, allergens (e.g., gluten and nut proteins), andmycotoxins.

Target analytes can include polypeptides (e.g., polymers of naturallyand/or non-naturally occurring amino acids such as Gly, Ala, Val, Leu,Ile, Pro, Phe, Trp, Cys, Met, Ser, Thr, Tyr, His, Lys, Arg, Asp, Glu,Asn, Gln, selenocysteine, ornithine, citrulline, hydroxyproline,methyllysine, carboxyglutamate), peptides, proteins, glycoproteins,lipoproteins; peptide-nucleic acids; hormones (such as peptide hormones(e.g., TRH and vasopressin), as well as synthetic and industrialpolypeptides.

In some embodiments, the compound of interest is a saturated or anunsaturated lipid, vitamin or polycyclic aromatic hydrocarbon. The term“saturated” as used herein refers to constituents that contain no doublebond in the acyl site of the molecule. The term “saturated lipid” asused herein refers to constituents of fats and oils that contain nodouble bond in the acyl chain sites of the molecule. The term“unsaturated” as used herein refers to constituents that contain one ormore sites of unsaturation in the molecule. The term “unsaturated lipid”as used herein refers to constituents of fats and oils that contain oneor more sites of unsaturation in the molecule. These may occur in thefatty acid portions of the molecule such as in triglycerides,phospholipids and glycolipids, or in alkyl chains in the molecule, suchas in carotenoids, hydrocarbons and fat soluble vitamins. The lipid canbe selected from the group consisting of saturated and unsaturated fattyacids, phospholipids, glycerolipids, glycerophospholipids,lysophosphoglycerolipids, sphingolipids, sterol lipids, prenol lipids,saccharolipids, carotenoids, waxes, and polyketides.

In another embodiment, the present disclosure can be used to retain,separate and resolve polycyclic aromatic hydrocarbon and relatedcompounds. Polycyclic aromatic hydrocarbons (PAHs) form a family ofnon-functionalised aromatic compounds composed of fused aromatic rings.There are about 2000 compounds classed as PAHs. PAHs and theirderivatives are widespread in the environment as a result of combustionprocesses, such as burning fossil fuels. They bind strongly to soilorganic matter (humic acids) and their rate of degradation in soil andother environmental compartments is usually slow. In addition, PAHsreaching watercourses are rapidly transferred to sediment.

PAHs are also formed during domestic and industrial combustion processessuch as extracting vegetable oils, herbs, spices and other foodmaterials, smoking and grilling food materials and the like. PAHs aretoxic and carcinogenic compounds. The control of their presence andlevels is of growing importance in the context of food safety and healthand safety regulations. Several of the effects of PAHs are enzymeinduction, immunosuppression, teratogenecity, and tumour promotion.

In particular embodiments, the lipid can be saturated or an unsaturatedfatty acid, monoacylglyceride, diacylglyceride, triacylglyceride,phospholipid or steroid. A triglyceride (TG, triacylglycerol, TAG, ortriacylglyceride) is an ester derived from glycerol and three fattyacids. As a blood lipid, they help enable the bidirectional transferenceof adipose fat and blood glucose from the liver. There are manytriglycerides: depending on the oil source, some are highly unsaturated,some less so. Triglycerides are the main constituents of vegetable oil(typically more unsaturated) and animal fats (typically more saturated).Triglycerides are a major component of human skin oils.

A steroid is a type of organic compound that contains a characteristicarrangement of four cycloalkane rings that are joined to each other.Examples of steroids include the dietary lipid cholesterol, the sexhormones estradiol and testosterone and the anti-inflammatory drugdexamethasone. The core of steroids is composed of seventeen carbonatoms bonded together that take the form of four fused rings: threecyclohexane rings and one cyclopentane ring. The steroids vary by thefunctional groups attached to this four-ring core and by the oxidationstate of the rings. Sterols are special forms of steroids, with ahydroxyl group at position-3 and a skeleton derived from cholestane.

Hundreds of distinct steroids are found in plants, animals and fungi.All steroids are made in cells either from the sterols lanosterol(animals and fungi) or from cycloartenol (plants). Both lanosterol andcycloartenol are derived from the cyclization of the triterpenesqualene.

In other embodiments, the compound of interest can be a fat solublevitamin selected from the group consisting of a vitamin C, vitamin B, orderivatives or combinations thereof. Fat-soluble vitamins are ofinterest because they are difficult to dissolve, often strong organicsolvents are necessary and separation via RPLC. Typically, fat-solublevitamins are separated using SFC conditions by the use of C18 silanes(ODS) or other alkyl bonded phases. These methods often utilize veryweak co-solvents and very high percentages of CO₂ in the mobile phase.One of the advantages of the present disclosure is the stationary phaseis modified with a pi-electron rich selector, e.g. 1-aminoanthracene.Coupling a pi-electron rich selector to the stationary phase maximizesthe chromatographic selectively between two critical pairs, such asvitamins D2 and D3, or K1 and K2.

Another advantage of the present disclosure is the stationary phaseshave selector with a relatively high pKa such that no acid or basicadditives are necessary to accomplish the separation. In one embodiment,the present disclosure relates to methodology utilizing a mobile phasewith no acid additive, basic additive, or both. In other embodiments,the present disclosure relates to methodology utilizing a mobile phasewith less than 5.0%, or 4.0%, or 3.0%, or 2.0%, 1.0%, or 0.5%, or 0.2%,or 0.1%, or 0.05% of an acid additive, basic additive, or both. Usingonly carbon dioxide modified with methanol as the co-solvent, a fast,generic, method can be created. For example, the separation of theafore-mentioned vitamin critical pairs is achieved using the stationaryphase of the present disclosure having, for example, 1-aminoanthraceneas the selector. In one embodiment, the level of resolution betweencritical pairs of Vitamins is higher than current methodology(resolution per same column length; use of sub-2 μm particles and 50 mmcolumn length).

The present disclosure can be used to retain, separate and resolveVitamin C and related compounds. Vitamin C[2-oxo-L-threo-hexono-1,4-lactone2,3-enediol] or L-ascorbic acid is awater-soluble vitamin and essential nutrient for humans. It is essentialin the formation of collagen, which is required for normal growth anddevelopment as well as tissue repair in all parts of the body. Vitamin Calso functions as an antioxidant that blocks the damage caused by freeradicals and directly reduces toxic chemicals and pollutants.

As humans do not produce vitamin C in the body, it is primarily obtainedfrom dietary sources such as fruits and vegetables. Lack of dietaryvitamin C may result in vitamin C deficiency. Severe vitamin Cdeficiency, also know as “scurvy,” leads to the formation of liver spotson skin, spongy gums, and bleeding from mucous membranes, or even death.

Currently, vitamin C is not only used as a dietary supplement, but alsoas an adjunct therapy for some viral infections and terminal cancers.The recommended daily intake of vitamin C for adults to preventdeficiency is 75 mg for females and 90 mg for males, both with atolerable upper level of 2,000 mg. For therapeutic usage indetoxification and cancer therapy, vitamin C is given intravenously atmuch higher doses. Although vitamin C toxicity is rare clinically,relatively high doses of oral intake may lead to stomach upset anddiarrhea. Assays for vitamin C blood levels have been developed and areused by patients and physicians to evaluate nutritional status or tooptimize therapeutic dosages. Measurement of these compounds are usefulindices of vitamin C nutritional status and the efficacy of certainvitamin C analogs.

In another embodiment, the present disclosure can be used to retain,separate and resolve Vitamin B and related compounds. B vitamins are agroup of water-soluble vitamins that play important roles in cellmetabolism. The B vitamins were once thought to be a single vitamin,referred to simply as vitamin B. Later research showed that they arechemically distinct vitamins that often coexist in the same foods. Ingeneral, supplements containing all eight are referred to as a vitamin Bcomplex. Individual B vitamin supplements are referred to by thespecific name of each vitamin (e.g., B1, B2, B3 etc.). A list of Bvitamins includes: Vitamin B1 (thiamine), Vitamin B2 (riboflavin),Vitamin B3 (niacin or niacinamide), Vitamin B5 (pantothenic acid),Vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine, or pyridoxinehydrochloride), Vitamin B7 (biotin), Vitamin B9 (folic acid), VitaminB12 (various cobalamins; commonly cyanocobalamin in vitaminsupplements).

The roles of the Vitamin B compound differ. For example, thiamine playsa central role in the generation of energy from carbohydrates. It isinvolved in RNA and DNA production, as well as nerve function. Itsactive form is a coenzyme called Thiamine pyrophosphate (TPP), whichtakes part in the conversion of pyruvate to acetyl Coenzyme A (CoA) inmetabolism. Riboflavin is involved in the energy production for theelectron transport chain, the citric acid cycle, as well as thecatabolism of fatty acids (beta oxidation). Niacin is composed of twostructures: nicotinic acid and nicotinamide. There are two co-enzymeforms of niacin: nicotinamide adenine dinucleotide (NAD) andnicotinamide adenine dinucleotide phosphate (NADP). Both play animportant role in energy transfer reactions in the metabolism ofglucose, fat and alcohol. NAD carries hydrogens and their electronsduring metabolic reactions, including the pathway from the citric acidcycle to the electron transport chain. NADP is a coenzyme in lipid andnucleic acid synthesis.

Pantothenic acid is involved in the oxidation of fatty acids andcarbohydrates. Coenzyme A, which can be synthesised from pantothenicacid, is involved in the synthesis of amino acids, fatty acids, ketones,cholesterol, phospholipids, steroid hormones, neurotransmitters (such asacetylcholine), and antibodies. Pyridoxine is usually stored in the bodyas pyridoxal 5′-phosphate (PLP), which is the co-enzyme form of vitaminB6. Pyridoxine is involved in the metabolism of amino acids and lipids;in the synthesis of neurotransmitters and hemoglobin, as well as in theproduction of nicotinic acid (vitamin B3). Pyridoxine also plays animportant role in gluconeogenesis. Biotin plays a key role in themetabolism of lipids, proteins and carbohydrates. It is a criticalco-enzyme of four carboxylases: acetyl CoA carboxylase, which isinvolved in the synthesis of fatty acids from acetate; propionyl CoAcarboxylase, involved in gluconeogenesis; β-methylcrotonyl Coacarboxylase, involved in the metabolism of leucin; and pyruvate CoAcarboxylase, which is involved in the metabolism of energy, amino acidsand cholesterol.

Folic acid acts as a co-enzyme in the form of tetrahydrofolate (THF),which is involved in the transfer of single-carbon units in themetabolism of nucleic acids and amino acids. THF is involved inpyrimidine nucleotide synthesis, so is needed for normal cell division,especially during pregnancy and infancy, which are times of rapidgrowth. Folate also aids in erythropoiesis, the production of red bloodcells. Vitamin B12 is involved in the cellular metabolism ofcarbohydrates, proteins and lipids. It is essential in the production ofblood cells in bone marrow, nerve sheaths and proteins. Vitamin B12functions as a co-enzyme in intermediary metabolism for the methioninesynthase reaction with methylcobalamin, and the methylmalonyl CoA mutasereaction with adenosylcobalamin.

The impact of any deficiency in these vitamins also differs. Vitamin B1thiamine Deficiency causes beriberi. Symptoms of this disease of thenervous system include weight loss, emotional disturbances, Wernicke'sencephalopathy (impaired sensory perception), weakness and pain in thelimbs, periods of irregular heartbeat, and edema (swelling of bodilytissues). Heart failure and death may occur in advanced cases. Chronicthiamine deficiency can also cause Korsakoffs syndrome, an irreversibledementia characterized by amnesia and compensatory confabulation.

Vitamin B2 riboflavin Deficiency causes ariboflavinosis. Symptoms mayinclude cheilosis (cracks in the lips), high sensitivity to sunlight,angular cheilitis, glossitis (inflammation of the tongue), seborrheicdermatitis or pseudo-syphilis (particularly affecting the scrotum orlabia majora and the mouth), pharyngitis (sore throat), hyperemia, andedema of the pharyngeal and oral mucosa.

Vitamin B3 niacin Deficiency, along with a deficiency of tryptophancauses pellagra. Symptoms include aggression, dermatitis, insomnia,weakness, mental confusion, and diarrhea. In advanced cases, pellagramay lead to dementia and death (the 3(+1) Ds: dermatitis, diarrhea,dementia, and death).

Vitamin B5 pantothenic acid Deficiency can result in acne andparesthesia, although it is uncommon. Vitamin B6 pyridoxine Deficiencymay lead to microcytic anemia (because pyridoxyl phosphate is thecofactor for heme synthesis), depression, dermatitis, high bloodpressure (hypertension), water retention, and elevated levels ofhomocysteine. Vitamin B7 biotin Deficiency does not typically causesymptoms in adults but may lead to impaired growth and neurologicaldisorders in infants. Multiple carboxylase deficiency, an inborn errorof metabolism, can lead to biotin deficiency even when dietary biotinintake is normal.

Vitamin B9 folic acid Deficiency results in a macrocytic anemia, andelevated levels of homocysteine. Deficiency in pregnant women can leadto birth defects. Supplementation is often recommended during pregnancy.Researchers have shown that folic acid might also slow the insidiouseffects of age on the brain. Vitamin B12 cobalamin Deficiency results ina macrocytic anemia, elevated homocysteine, peripheral neuropathy,memory loss and other cognitive deficits. It is most likely to occuramong elderly people, as absorption through the gut declines with age;the autoimmune disease pernicious anemia is another common cause. It canalso cause symptoms of mania and psychosis. In rare extreme cases,paralysis can result. Measurement of these compounds are useful indicesof vitamin B nutritional status and the efficacy of certain vitamin Banalogs.

In other embodiments, the compound of interest can be a fat solublevitamin selected from the group consisting of a vitamin D, vitamin A,vitamin K, vitamin E, betacarotene, or derivatives or combinationsthereof. The present disclosure can be used to retain, separate andresolve Vitamin D and related compounds. Vitamin D is an essentialnutrient with important physiological roles in the positive regulationof calcium homeostasis. Vitamin D can be made de novo in the skin byexposure to sunlight or it can be absorbed from the diet. There are twoforms of vitamin D; vitamin D2 (ergo calciferol) and vitamin D3(cholecalciferol). Vitamin D3 is the form synthesized de novo byanimals. It is also a common supplement added to milk products andcertain food products produced in the United States. Both dietary andintrinsically synthesized vitamin D3 must undergo metabolic activationto generate the bioactive metabolites. In humans, the initial step ofvitamin D3 activation occurs primarily in the liver and involveshydroxylation to form the intermediate metabolite25-hydroxycholecalciferol (calcifediol), which is enzymaticallyhydroxylated at the 25 position. Calcifediol is the major form ofVitamin D3 in the circulation. Circulating calcifediol is then convertedby the kidney to form 1,25-dihydroxyvitamin D3 (calcitriol), which isgenerally believed to be the metabolite of Vitamin D3 with the highestbiological activity. Vitamin D2 is derived from fungal and plantsources. Many over-the-counter dietary supplements containergocalciferol (vitamin D2) rather than cholecalciferol (vitamin D3).Drisdol, the only high-potency prescription form of vitamin D availablein the United States, is formulated with ergocalciferol. Vitamin D2undergoes a similar pathway of metabolic activation in humans as vitaminD3, forming the metabolites calcifediol and calcitriol. Vitamin D2 andvitamin D3 have long been assumed to be biologically equivalent inhumans, however recent reports suggest that there may be differences inthe bioactivity and bioavailability of these two forms of vitamin D.Measurement of these compounds are useful indices of vitamin Dnutritional status and the efficacy of certain vitamin D analogs.

In another embodiment, the present disclosure can be used to retain,separate and resolve Vitamin A and related compounds. Vitamin A is agroup of unsaturated nutritional organic compounds, that includesretinol, retinal, retinoic acid, and several provitamin A carotenoids,among which beta-carotene is the most important. Vitamin A has multiplefunctions: it is important for growth and development, for themaintenance of the immune system and good vision. Vitamin A is needed bythe retina of the eye in the form of retinal, which combines withprotein opsin to form rhodopsin the light-absorbing molecule, that isnecessary for both low-light (scotopic vision) and color vision. VitaminA also functions in a very different role as an irreversibly oxidizedform of retinol known as retinoic acid, which is an importanthormone-like growth factor for epithelial and other cells.

In foods of animal origin, the major form of vitamin A is an ester,primarily retinyl palmitate, which is converted to retinol (chemicallyan alcohol) in the small intestine. The retinol form functions as astorage form of the vitamin, and can be converted to and from itsvisually active aldehyde form, retinal. The associated acid (retinoicacid), a metabolite that can be irreversibly synthesized from vitamin A,has only partial vitamin A activity, and does not function in the retinafor the visual cycle. Retinoic acid is used for growth and cellulardifferentiation.

All forms of vitamin A have a beta-ionone ring to which an isoprenoidchain is attached, called a retinyl group. Both structural features areessential for vitamin activity. The orange pigment ofcarrots—beta-carotene—can be represented as two connected retinylgroups, which are used in the body to contribute to vitamin A levels.Alpha-carotene and gamma-carotene also have a single retinyl group,which give them some vitamin activity.

Vitamin A can be found in two principal forms in foods: (i) Retinol, theform of vitamin A absorbed when eating animal food sources, is a yellow,fat-soluble substance. Since the pure alcohol form is unstable, thevitamin is found in tissues in a form of retinyl ester. It is alsocommercially produced and administered as esters such as retinyl acetateor palmitate. (ii) The carotenes alpha-carotene, beta-carotene,gamma-carotene; and the xanthophyll beta-cryptoxanthin (all of whichcontain beta-ionone rings), but no other carotenoids, function asprovitamin A in herbivores and omnivore animals, which possess theenzyme (15-15′-dioxygenase) which cleaves beta-carotene in theintestinal mucosa and converts it to retinol. In general, carnivores arepoor converters of ionone-containing carotenoids, and pure carnivoressuch as cats and ferrets lack 15-15′-dioxygenase and cannot convert anycarotenoids to retinal (resulting in none of the carotenoids being formsof vitamin A for these species). Measurement of these compounds areuseful indices of vitamin A nutritional status and the efficacy ofcertain vitamin A analogs.

In another embodiment, the present disclosure can be used to retain,separate and resolve Vitamin K and related compounds. Vitamin K is agroup of structurally similar, fat-soluble vitamins that the human bodyneeds for post-translational modification of certain proteins requiredfor blood coagulation, and in metabolic pathways in bone and othertissue. They are 2-methyl-1,4-naphthoquinone (3-) derivatives. Thisgroup of vitamins includes two natural vitamers: vitamin K1 and vitaminK2.

Vitamin K1, also known as phylloquinone, phytomenadione, orphytonadione, is synthesized by plants, and is found in highest amountsin green leafy vegetables because it is directly involved inphotosynthesis. It may be thought of as the “plant form” of vitamin K.It is active in animals and may perform the classic functions of vitaminK in animals, including its activity in the production of blood-clottingproteins. Animals may also convert it to vitamin K2.

Vitamin K2, the main storage form in animals, has several subtypes,which differ in isoprenoid chain length. These vitamin K2 homologues arecalled menaquinones, and are characterized by the number of isoprenoidresidues in their side chains. Menaquinones are abbreviated MK-n, whereM stands for menaquinone, the K stands for vitamin K, and the nrepresents the number of isoprenoid side chain residues. For example,menaquinone-4 (abbreviated MK-4) has four isoprene residues in its sidechain. Menaquinone-4 (also known as menatetrenone from its four isopreneresidues) is the most common type of vitamin K2 in animal products sinceMK-4 is normally synthesized from vitamin K1 in certain animal tissues(arterial walls, pancreas, and testes) by replacement of the phytyl tailwith an unsaturated geranylgeranyl tail containing four isoprene units,thus yielding menaquinone-4. This homolog of vitamin K2 may have enzymefunctions that are distinct from those of vitamin K1.

Bacteria in the colon (large intestine) can also convert K1 into vitaminK2. In addition, bacteria typically lengthen the isopreneoid side chainof vitamin K2 to produce a range of vitamin K2 forms, most notably theMK-7 to MK-11 homologues of vitamin K2. All forms of K2 other than MK-4can only be produced by bacteria, which use these forms in anaerobicrespiration. The MK-7 and other bacteria-derived form of vitamin K2exhibit vitamin K activity in animals, but MK-7's extra utility overMK-4, if any, is unclear and is presently a matter of investigation.

Three synthetic types of vitamin K are known: vitamins K3, K4, and K5.Although the natural K1 and all K2 homologues have proven nontoxic, thesynthetic form K3 (menadione) has shown toxicity. K4, and K5 are alsonon toxic. Measurement of these compounds are useful indices of vitaminK nutritional status and the efficacy of certain vitamin K analogs.

In another embodiment, the present disclosure can be used to retain,separate and resolve Vitamin E and related compounds. Vitamin E refersto a group of eight fat-soluble compounds that include both tocopherolsand tocotrienols. Of the many different forms of vitamin E, γ-tocopherolis the most common in the North American diet. γ-Tocopherol can be foundin corn oil, soybean oil, margarine, and dressings. α-tocopherol, themost biologically active form of vitamin E, is the second-most commonform of vitamin E in the diet. This variant can be found most abundantlyin wheat germ oil, sunflower, and safflower oils. As a fat-solubleantioxidant, it stops the production of reactive oxygen species formedwhen fat undergoes oxidation. Amounts over 1,000 mg (1,500 IU) per dayare called Hypervitaminosis E, as they may increase the risk of bleedingproblems and vitamin K deficiency. Measurement of these compounds areuseful indices of vitamin e nutritional status and the efficacy ofcertain vitamin E analogs.

In general, a sample is a composition including at least one targetanalyte (e.g., an analyte of the class or kind disclosed above, togetherwith a matrix). Samples can include a solid, liquid, gas, mixture,material (e.g., of intermediary consistency, such as an extract, cell,tissue, organisms) or a combination thereof. In various embodiments, thesample is a bodily sample, an environmental sample, a food sample, asynthetic sample, an extract (e.g., obtained by separation techniques),or a combination thereof.

Bodily samples can include any sample that is derived from the body ofan individual. In this context, the individual can be an animal, forexample a mammal, for example a human. Other example individuals includea mouse, rat, guinea-pig, rabbit, cat, dog, goat, sheep, pig, cow, orhorse. The individual can be a patient, for example, an individualsuffering from a disease or being suspected of suffering from a disease.A bodily sample can be a bodily fluid or tissue, for example taken forthe purpose of a scientific or medical test, such as for studying ordiagnosing a disease (e.g., by detecting and/or identifying a pathogenor the presence of a biomarker). Bodily samples can also include cells,for example, pathogens or cells of the individual bodily sample (e.g.,tumour cells). Such bodily samples can be obtained by known methodsincluding tissue biopsy (e.g., punch biopsy) and by taking blood,bronchial aspirate, sputum, urine, faeces, or other body fluids.Exemplary bodily samples include humour, whole blood, plasma, serum,umbilical cord blood (in particular, blood obtained by percutaneousumbilical cord blood sampling (PUBS), cerebrospinal fluid (CSF), saliva,amniotic fluid, breast milk, secretion, ichor, urine, faeces, meconium,skin, nail, hair, umbilicus, gastric contents, placenta, bone marrow,peripheral blood lymphocytes (PBL), and solid organ tissue extract.

Environmental samples can include any sample that is derived from theenvironment, such as the natural environment (e.g., seas, soils, air,and flora) or the manmade environment (e.g., canals, tunnels,buildings). Exemplary environmental samples include water (e.g.,drinking water, river water, surface water, ground water, potable water,sewage, effluent, wastewater, or leachate), soil, air, sediment, biota(e.g., soil biota), flora, fauna (e.g., fish), and earth mass (e.g.,excavated material).

Food samples can include any sample that is derived from food (includingbeverages). Such food samples can be used for various purposesincluding, for example, (1) to check whether a food is safe; (2) tocheck whether a food contained harmful contaminants at the time the foodwas eaten (retained samples) or whether a food does not contain harmfulcontaminants; (3) to check whether a food contains only permittedadditives (e.g., regulatory compliance); (4) to check whether itcontains the correct levels of mandatory ingredients (e.g., whether thedeclarations on the label of the food are correct); or (5) to analyzethe amounts of nutrients contained in the food. Exemplary food samplesinclude edible products of animal, vegetable or synthetic origin (e.g.,milk, bread, eggs, or meat), meals, drinks, and parts thereof, such asretain samples. Food samples can also include fruits, vegetables,pulses, nuts, oil seeds, oil fruits, cereals, tea, coffee, herbalinfusions, cocoa, hops, herbs, spices, sugar plants, meat, fat, kidney,liver, offal, milk, eggs, honey, fish, and beverages.

Synthetic samples can include any sample that is derived from anindustrial process. The industrial process can be a biologicalindustrial process (e.g., processes using biological material containinggenetic information and capable of reproducing itself or beingreproduced in a biological system, such as fermentation processes usingtransfected cells) or a non-biological industrial process (e.g., thechemical synthesis or degradation of a compound such as apharmaceutical). Synthetic samples can be used to check and monitor theprogress of the industrial process, to determine the yield of thedesired product, and/or measure the amount of side products and/orstarting materials.

EXAMPLES

Materials

All reagents were used as received unless otherwise noted. Those skilledin the art will recognize that equivalents of the following supplies andsuppliers exist and, as such, the suppliers listed below are not to beconstrued as limiting.

Characterization Techniques

Those skilled in the art will recognize that equivalents of thefollowing instruments and suppliers exist and, as such, the instrumentslisted below are not to be construed as limiting.

The % C values were measured by combustion analysis (CE-440 ElementalAnalyzer; Exeter Analytical Inc., North Chelmsford, Mass.) or byCoulometric Carbon Analyzer (modules CM5300, CM5014, UIC Inc., Joliet,Ill.). Bromine and Chlorine content were determined by flask combustionfollowed by ion chromatography (Atlantic Microlab, Norcross, Ga.). Thespecific surface areas (SSA), specific pore volumes (SPV) and theaverage pore diameters (APD) of these materials were measured using themulti-point N₂ sorption method (Micromeritics ASAP 2400; MicromeriticsInstruments Inc., Norcross, Ga.). The SSA was calculated using the BETmethod, the SPV was the single point value determined for P/P₀>0.98 andthe APD was calculated from the desorption leg of the isotherm using theBJH method. The micropore surface area (MSA) was determined as thecumulative adsorption pore diameter data for pores <34 Å subtracted fromthe specific surface area (SSA). The median mesopore diameter (MMPD) andmesopore pore volume (MPV) were measured by Mercury Porosimetry(Micromeritics AutoPore II 9220 or AutoPore IV, Micromeritics, Norcross,Ga.). Skeletal densities were measured using a Micromeritics AccuPyc1330 Helium Pycnometer (V2.04N, Norcross, Ga.). Particle sizes weremeasured using a Beckman Coulter Multisizer 3 analyzer (30 μm aperture,70,000 counts; Miami, Fla.). The particle diameter (dp₅₀) was measuredas the 50% cumulative diameter of the volume based particle sizedistribution. The width of the distribution was measured as the 90%cumulative volume diameter divided by the 10% cumulative volume diameter(denoted 90/10 ratio). Viscosity was determined for these materialsusing a Brookfield digital viscometer Model DV-II (Middleboro, Mass.).Measurements of pH were made with an Oakton pH100 Series meter(Cole-Palmer, Vernon Hills, Ill.) and were calibrated using ORION®(Thermo Electron, Beverly, Mass.) pH buffered standards at ambienttemperature immediately before use. Titrations were performed using aMETROHM® 716 DMS TITRINO® autotitrator (Metrohm, Hersau, Switzerland),and are reported as milliequivalents per gram (mequiv/g). Coveragelevels for the epoxide were determined by titrating the OH⁻ liberatedupon addition of sodium thiosulfate. Multinuclear (¹³C, ²⁹Si) CP-MAS NMRspectra were obtained using a Bruker Instruments Avance-300 spectrometer(7 mm double broadband probe). The spinning speed was typically 5.0-6.5kHz, recycle delay was 5 sec. and the cross-polarization contact timewas 6 msec. Reported ¹³C and ²⁹Si CP-MAS NMR spectral shifts wererecorded relative to tetramethylsilane using the external standardsadamantane (¹³C CP-MAS NMR, δ 38.55) and hexamethylcyclotrisiloxane(²⁹Si CP-MAS NMR, δ −9.62). Populations of different siliconenvironments were evaluated by spectral deconvolution using DMFitsoftware. [Massiot, D.; Fayon, F.; Capron, M.; King, I.; Le Calvé, S.;Alonso, B.; Durand, J.-O.; Bujoli, B.; Gan, Z.; Hoatson, G. Magn. Reson.Chem. 2002, 40, 70-76]

Example 1 Preparation of Epoxide Layer for Stationary Phases

In a typical reaction, hybrid porous particles were dispersed in asolution of glycidoxypropyltrimethoxysilane/methanol (0.25 mL/g) (GLYMO,Aldrich, Milwaukee, Wis.,) in a 20 mM acetate buffer (pH 5.5, preparedusing acetic acid and sodium acetate, J.T. BAKER® 5 mL/g dilution) thathad be premixed at 70° C. for 60 minutes. The mixture was held at 70° C.for 20 hours. The reaction was then cooled and the product was filteredand washed successively with water and methanol (J.T. BAKER®). Theproduct was then dried at 80° C. under reduced pressure for 16 hours.The specific particles used are shown in Table 1.

TABLE 1 Specific Base Particles Utilized Entry Material B1 HybridOrganic Silica (3.8 μm, 90 {acute over (Å)} APD, 1.3 cm³/g TPV)¹ B2Hybrid Organic Silica (3.8 μm, 115 {acute over (Å)} APD, 1.3 cm³/g TPV)¹B3 Hybrid Organic Silica (2.3 μm, 115 {acute over (Å)} APD, 1.3 cm³/gTPV)¹ B4 Hybrid Organic Silica (1.7 μm, 143 {acute over (Å)} APD, 0.73cm³/g TPV B5 Hybrid Organic Silica (1.7 μm, 106 {acute over (Å)} APD,1.25 cm³/g TPV ¹As described in U.S. Pat. No. 7,919,177, U.S. Pat. No.7,223,473, U.S. Pat. No. 6,686,035 and WO2011084506

Reaction data is listed in Table 2. Specific changes to this generalprocedure include: 1) Material 1E was prepared utilizing a 6 hourreaction time, 2) Material 1F was prepared utilizing 100 mM acetatebuffer, 3) Material 1G was prepared utilizing a 50° C. hold for 20hours, 4) Material 1H was prepared utilizing a 50° C. premix and 50° C.hold. Total surface coverages of 3.90-6.0 μmol/m² were determined by thedifference in particle % C before and after the surface modification asmeasured by elemental analysis. Analysis of these materials by ¹³CCP-MAS NMR spectroscopy indicates a mixture of epoxy and diol groups arepresent for these materials.

TABLE 2 Coverage of initial layer of Stationary Phases Coverage Total ofBase Material GPTMS Coverage¹ epoxide² Example Particle Amount (g)Amount (g) (μmol/m²) (μmol/m²) 1A B1 70 90.7 3.90 1.52 1B B2 40 39.04.80 1.86 1C B3 25 23.4 4.76 1.76 1D B2 20 17.3 4.71 1.67 1E B2 60 52.14.73 3.11 1F B2 20 19.3 4.87 1.51 1G B2 20 19.3 5.10 2.38 1H B2 20 19.34.22 3.23 1I B4 30 12.9 4.23 1.15 1J B4 60 39.4 5.01 1.88 1K B4 60 52.55.94 2.67 1L B5 20 20.0 5.05 1.82 ¹This refers to the combined coveragefrom the bonded GPTMS silane - coverage from unhydrolyzed epoxides +coverage from hydrolyzed epoxides (as the diol). ²As determined bytitration.

Example 2 Preparation of Stationary Phases with Diolfunctionality

In a typical reaction, hybrid porous particles were dispersed in asolution of glycidoxypropyltrimethoxysilane/methanol (0.25 mL/g) (GLYMO,Aldrich, Milwaukee, Wis.) in a 20 mM acetate buffer (pH 5.5, preparedusing acetic acid and sodium acetate, J.T. BAKER® 5 mL/g dilution) thathad be premixed at 70° C. for 60 minutes. The mixture was held at 70° C.for 20 hours. The reaction was then cooled and the product was filteredand washed successively with water and methanol (J.T. BAKER®). Thematerial was then refluxed in a 0.1 M acetic acid solution (5 mL/gdilution, J.T. BAKER®) at 70° C. for 20 hours. The reaction was thencooled and the product was filtered and washed successively with waterand methanol (J.T. BAKER®). The product was then dried at 80° C. underreduced pressure for 16 hours. Reaction data is listed in Table 3.Surface coverages of 0.93-6.0 μmol/m² were determined by the differencein particle % C before and after the surface modification as measured byelemental analysis. Analysis of these materials by ¹³C CP-MAS NMRspectroscopy indicates no measurable amount of epoxide groups remain,having only diol groups present for these materials.

TABLE 3 Coverage of initial layer of Stationary Phases Coverage Total ofBase Material GPTMS Coverage¹ epoxide² Example Particle Amount (g)Amount (g) (μmol/m²) (μmol/m²) 2A B3 20 4.9 2.71 N/A 2B B3 20 7.0 3.37N/A 2C B1 70 90.3 3.16 N/A 2D B1 20 7.3 1.76 N/A 2E B1 20 4.4 0.93 N/A2F B5 50 48.6 4.91 N/A

Example 3—Preparation of Stationary Phases with Mixed Functionality

In a standard experiment, 10 g of a material prepared above weredispersed in a solvent such as, but not limited to water, iso-propanol,or dioxane. An amount of nucleophile in excess of the epoxide coveragedetermined for the material prepared above was added and the mixtureheated to 70° C. for 16 hours. Table 4 provides the list of specificnucleophiles used. After reaction, the particles were washedsuccessively with water and 0.5M acetic acid, and the material was thenstirred in a 0.1 M acetic acid solution (5 mL/g dilution, J.T. BAKER®)at 70° C. for 20 hours. The reaction was then cooled and the product wasfiltered and washed successively with water and methanol (J.T. BAKER®).The product was then dried at 80° C. under reduced pressure for 16hours. Reaction data is listed in Table 5. Nucleophile surfaceconcentrations of 0.2-2.3 mol/m² were determined by the difference inparticle % C, % N or % S before and after the surface modification asmeasured by elemental analysis. Analysis of these materials by ¹³CCP-MAS NMR spectroscopy indicates no measurable amount of epoxide groupsremain.

TABLE 4 List of Specific Nucleophiles Utilized Entry Nucleophile N11-aminoanthracene N2 4-n-octylaniline N3 6-aminoquinoline N4 aniline N51-naphthylamine N6 8-aminoquinoline N7 2-aminoanthracene N8 benzylamineN9 2-picolylamine N10 pyridine N11 N-octadecylamine N12 diethylamine

TABLE 5 Base Material Total Representative Coverage NucleophilePreparation from Initial Surface from Bonding Nucleophile ConcentrationExample Example 1 (μmol/m²) Used (μmol/m²) 3A 1C 4.57 N1 1.61 3B 1I 4.23N1 0.98 3C 1J 5.01 N1 1.16 3D 1K 5.94 N1 1.95 3E 1H 3.82 N2 2.01 3F 1C4.58 N3 1.10 3G 1C 4.58 N4 1.53 3H 1C 4.58 N5 1.65 3I 1C 4.66 N6 1.48 3J1C 4.35 N7 0.66 3K 1C 4.39 N8 1.21 3L 1L 5.05 N9 1.32 3M 1C 4.66 N101.07 3N 1H 4.62 N11 2.28 3O 1L 5.05 N12 1.60 3P 1C 4.35 N1 1.41

Example 4 Further Characterization of Stationary Phases

The general procedure for bondings/functionalization of particles thatis detailed in Examples 1-3 is applied to modify the surface silanolgroups of different porous materials. Included in this are monolithic,spherical, granular, superficially porous and irregular materials thatare silica, hybrid inorganic/organic materials, hybrid inorganic/organicsurface layers on hybrid inorganic/organic, silica, titania, alumina,zirconia, polymeric or carbon materials, and silica surface layers onhybrid inorganic/organic, silica, titania, alumina, zirconia orpolymeric or carbon materials. Also includes are stationary phasematerials in the form of a spherical material, non-spherical material(e.g., including toroids, polyhedron); stationary phase materials havinga highly spherical core morphology, a rod shaped core morphology, abent-rod shaped core morphology, a toroid shaped core morphology; or adumbbell shaped core morphology; and stationary phase materials having amixture of highly spherical, rod shaped, bent rod shaped, toroid shaped,or dumbbell shaped morphologies. Example hybrid materials are shown inU.S. Pat. Nos. 4,017,528, 6,528,167, 6,686,035, and 7,175,913 as well asInternational Publication No. WO2008/103423, the contents of which arehereby incorporated by reference in their entireties. Superficiallyporous particle include those describe in U.S. Pub. Nos. 2013/0112605,2007/0189944, and 2010/061367, the contents of which are herebyincorporated by reference in their entireties. The particles size forspherical, granular or irregular materials can vary from 5-500 μm; morepreferably 15-100 μm; more preferably 20-80 μm; more preferably 40-60am. The APD for these materials can vary from 30 to 2,000 Å; morepreferably 40 to 200 Å; more preferably 50 to 150 Å. The SSA for thesematerials can vary from 20 to 1000 m²/g; more preferably 90 to 800 m²/g;more preferably 150 to 600 m²/g; more preferably 300 to 550 m²/g. TheTPV for these materials can vary from 0.3 to 1.5 cm³/g; more preferably0.5 to 1.4 cm³/g; more preferably 0.7 to 1.3 cm³/g. The macroporediameter for monolithic materials can vary from 0.1 to 30 am, morepreferably 0.5 to 25 am, more preferably 1 to 20 am.

Example 5 Stationary Phases Show Minimal Analyte Retention VariationOver Time Under Chromatographic Conditions

The average % retention change was calculated by taking the percentdifference of the average absolute peak retentions measured from the day3, 10 or 30 chromatographic tests from the average absolute peakretentions measured on the day one chromatographic test. For each daytested, the columns were equilibrated under Mix1 test conditions for 20minutes followed by three injections of Mix1 and then equilibrated underMix2 Test conditions for 10 minutes, followed by three injections ofMix2. Conditions are shown in Table 6. Results are shown in Tables 7 and8.

The % Less Retention was calculated by taking the percent difference ofthe day one average absolute peak retentions measured for Mix 1 and Mix2 from the day one average absolute peak retentions measured for Mix 1and Mix 2 on example 1 Å.

TABLE 6 Chromatographic Test Conditions for Retention ChangeMeasurements Co-Solvent Mix1 5% methanol Sample Mix1 3-benzoylpyridine(0.1 mg/mL) Co-Solvent Mix2 10% methanol Sample Mix2 caffeine, thymine,papaverine, prednisolone, sulfanilamide (0.2 mg/mL each) ColumnDimension 2.1 × 150 mm Flow Rate 1.0 mL/min Column Temperature 50° C.Back Pressure 1800 psi Detector ACQUITY ® PDA with SFC Flow CellDetector Setting 254 nm 40 spec/sec Weak Needle Wash iso-propanolInjection 1.0 μL (2.0 μL loop with PLUNO injection mode) InstrumentUPC² ® Software Empower

TABLE 7 Retention Change from Example 2 Materials Over Time Average %Retention Change Example 3 Day Test 10 Day Test 30 Day Test 2A 0.0 / /2B 0.2 / / 2C 0.2 / 1.8 2D 3.1 / 1.2 2E 0.4 / 0.2 / indicates that thistest was not performed for this material.

TABLE 8 Retention Change From Comparable Materials Over Time Average %Retention Change Example 3 Day Test 10 Day Test 30 Day Test 8-1 0.8 / /8-2 0.0 0.6 0.7 8-3 0.2 1.6 / 8-4 0.7 2.0 / 8-5 0.2 0.6 / 8-6 0.4 2.0 /8-7 1.3 2.2 / 8-8 1.0 1.6 0.4 8-9 0.5 0.0 1.1 8-10 2.4 1.1 1.0 8-11 0.22.3 2.9 / indicates that this test was not performed for this material.

Example 6 Surface Functionalization of Organic-Inorganic HybridParticles with Glycidoxypropyltrimethoxysilane (GPTMS) and1-Aminoanthracene

The structures of GPTMS and 1-aminoanthracene are shown in FIG. 1. FIG.1A shows the structure of GPTMS, a silane surface modifier. Part 105shows the surface reactive group of GPTMS (trialkoxysilane), while part110 shows the reactive group (epoxide). FIG. 1B shows the selectivityligand (1-aminoanthracene).

The GPTMS is first allowed to pre form oligomers by pre incubation at70° C. in 20 mM sodium Acetate buffer pH 5.0. During incubation smalloligomers of the hydrolyzed silanes are formed. After a suitable preincubation period the particles to be modified are added as a drypowder. The oligomers and any remaining monomer react with the materialsurface to produce a high surface coverage of silane modifier covalentlyattached to the material surface as shown in FIG. 2.

FIG. 2 shows a reaction of the silane coupling agent with theorganic-inorganic hybrid material surface. The silane (205) is depictedas a monomer for simplicity. Pre formed oligomeric silanes can couple inthe same manner as the surface reactive group (210). In someembodiments, a portion of the silanes not attached to the surface canalso couple to the coating (e.g., cross polymerization). Under reactionconditions, methanol is lost (215) to give surface modified particles(220).

After the silane has reacted with the chromatographic material, excessreagents and buffer salts are removed by washing with MILLI-Q® water andthe materials are transferred into an organic solvent (e.g.,1,4-dioxane) and the 1-aminoanthracene added. The amino group of the1-aminoanthracene couples to the surface through the pendant epoxidegroups of the GPTMS. The proportion of epoxy groups converted can becontrolled by limiting the quantity of 1-aminoanthracene added. Thecoupled materials are then washed into 0.5M acetic acid and theunreacted epoxide groups hydrolyzed to the corresponding diol as shownin FIG. 3.

The resulting 1-aminoanthracene/diol surface contains uniformlydistributed 1-aminoanthracene groups and provides excellent selectivitywhile the diol shields the surface silanols from interaction withanalyte. The multicomponent surface is superior to the singular diolsurface or the singular 1-aminoanthracene surface.

FIG. 3 shows reaction of the surface modified particle (305) with aselectivity ligand (310). The reaction conditions (315) are given andinclude treatment with isopropanol and 0.5 M acetic acid at 70° C. Theresult is a stationary phase particle with a multicomponent surface forchromatographic separation (320).

Alternatively, the multicomponent surface can be produced underconditions which create a polymerized surface by using a silane couplingagent with a pendant reactive group as the bonded phase under reactionconditions that simultaneously bond to the base material surface,partially react the pendant reactive group to form inert pendant groupsand also produce limited polymerization between pendant reactive groupson adjacent silane coupling agent molecules.

Similarly, the multicomponent surface can be produced under conditionswhich create a polymerized surface by covalently bonding a secondchemical agent capable of interacting with an analyte to affectretention through introduction of charged, uncharged, polar, nonpolar,lipophilic or hydrophilic character to the chromatographic phase.Alternatively, under certain conditions the epoxy groups of GPTMS canreact with the hydroxyl groups of adjacent silanes to form ether bridgeswhich crosslink the GPTMS on the surface. Such cross linking can providestability to the bonded phase and can also enhance silanol shielding.The existence of such cross links is consistent with NMR analysis ofthese materials. An embodiment of the types of crosslinked surface isshown in FIG. 4. FIG. 4 also show a crosslinked silane group in thecoating wherein the silane is not attached to the surface. Such astructure would demonstrate the formation of ether bridges throughpolymerization of the surface epoxides.

Example 7

FIG. 5 shows two potential synthetic routes to preparing achromatographic stationary phase of the present disclosure. As shown inthe scheme (500), an unmodified BEH particle (505) can be chemicallymodified in at least two different ways. Accordingly, one option, achemical modifying agent (510) is first prepared by reacting GPTMS with1-aminoanthracene. Agent 510 is then reacted with the BEH particle (505)to give a functionalized chromatographic surface (515).

Alternatively, FIG. 5 shows a different synthetic route. In thisembodiment, particle 505 is reacted directly with GPTMS (512) to give aGPTMS-modified surface (520). Surface 520 can then be reacted with1-aminoanthracene (525) to give a functionalized chromatographic surface(515).

In a preferred embodiment, the second reaction pathway comprising firstreacting particle 505 with GPTMS (512) followed by functionalizationwith 1-aminoanthracene (525) is performed over the first reactionpathway comprising reacting particle 505 with chemical modifying agent510, which can be a pre-formed chemical modifier.

Example 8 GPTMS Bonding on Organic-Inorganic Hybrid Mitigates RetentionDrift or Change

As shown in FIG. 6, treatment of a bridged ethylene hybrid (BEH)stationary phase with glycidoxypropyltrimethoxysilane (GPTMS) followedby a subsequent epoxide opening reaction to give a diol cansignificantly mitigate the effects of retention drift. Graph 600 shows aplot of the % Original Retention of unfunctionalized 3 μm BEH particles(605) compared with diol-functionalized 3 μm BEH particles (610). The %original retention is given as a function of time, with days on the xaxis. The results indicate that, in at least some preferred embodiments,functionalization of a chromatographic surface with GPTMS and subsequentepoxide opening to give a diol can mitigate retention drift. The resultsalso show that the GPTMS coating alone addresses the issue of retentiondrift and also provides significant retention.

Example 9 Separation of Compounds of Interest Using 1-AminoanthraceneBased Stationary Phase

Using simple chromatographic conditions, the methodology and stationaryphases of the present disclosure provides superior separation ofcompounds of interest, e.g. lipids and fat-soluble Vitamins, compared tocurrent, state-of-the-art methodology. They also deliver differentselectivity, as well, which is advantageous for revolving differentclasses of compounds.

An ACQUITY UPC²® system equipped with: Convergence Chromatography BinarySolvent Manager (ccBSM), Sample Manager (SM), Convergence ChromatographyManager (CCM), Column Manager (CM-A), and Photodiode Array (PDA) wasused. The system and separation conditions are provided as follows:

First conditions: Flow Rate: Gradient: 3-20% methanol in CO₂ in 2 mins;20% methanol in CO2 for 0.5 mins; 20-3% methanol in CO₂ in 0.5 mins.ABPR setting: 2175 psi. Column Temperature: 40° C. Detection: 235 nmcompensated 400-500 nm (320 nm compensated 400-500 nm for vitamin A, asneeded). Column Dimensions: 3.0×50 mm. Stationary Phase:1-aminoanthracene modified diol, 2.5 μm as provided in Example 7.

Second conditions: Flow Rate: 2.0 mL/min. Gradient: 3-8% methanol in CO₂in 3 mins (#8 curve); 8-35% methanol in CO₂ in 0.5 mins (#1 curve); 35%methanol in CO₂ for 1 minute; 35-3% methanol in CO₂ in 0.5 mins (#1curve). ABPR:1800 psi. Column Temperature: 50° C. Sample: GLC85 lipidsstandard (Nu-chek Prep, Elysian, Minn., USA), 1 g/L in CHCl₃ stockdiluted 20× with 1:1 heptane:IPA. Column Dimensions: 3.0×50 mm.Stationary Phase: 1-aminoanthracene modified diol, 2.5 μm as provided inExample 7.

Peak detection and structural determination was also provided using anACQUITY® SQD2 Mass Spectrometer: 3.46 kV capillary; 25V cone; 350° C.source; 500 L/hr desolvation gas; 10 L/hr cone gas; LM Res 11.8; HM Res15.1; −0.2 Ion Energy.

FIGS. 7-16 show lipid separations achieved using the methodology andstationary phases of the present disclosure. The results of theseseparations show that 1-aminoanthracene coupling is exceptional atretaining and separating fat-soluble vitamins, lipids and metabolites.The 1-aminoanthracene coupling enhances the shape/isomeric selectivitycompared to C18 bonded phases.

These results are surprising because the mixing of a hydrophobic andhydrophilic groups on the same ligand is not only challenging, but anatypical motif for stationary phases. Oftentimes, pi-electron richligands are used for very specialized separations and used to giveslightly different selectivity from C18 bonded phases. It was found thatvery different selectivity exists between 1-aminoanthracene basedstationary phase and, for example, an ACQUITY UPC²® HSS C18 SB (used forcomparison purposes in the present disclosure).

In the case of fat-soluble vitamins, as their name suggests, thesemolecules are nonpolar and often have few ionizable groups. In someembodiments, the stationary phase can contain residual amines which maynot enhance the separation of nonpolar compounds. In some embodiments,these groups can control the surface pH of the ligand. The presence ofthese groups would not indicate the separation of fat soluble vitaminsis possible. Usually, vitamins, lipid and metabolite separations areperformed on C18 bonded phases, such as ACQUITY UPC²® HSS C18 SB. Onthat material, alkyl chains are the retention selectors resulting inhigh methylene/hydrophobic selectivity, but little shape/isomericselectivity. Materials of the present disclosure, such as thosecontaining 1-aminoanthracene, are exceptional at retaining andseparating fat-soluble vitamins, lipids and metabolites. These materialsalso enhance the shape/isomeric selectivity as compared to C18 bondedphases.

Working with lipids resulted in a similar conclusion that the1-aminoanthracene coupling is superior to ACQUITY UPC²® HSS C18 SB. Whenusing optimized methods, more lipid peaks are resolved on the1-aminoanthracene coupled prototype. The increased number of peaksobserved is related to the selectivity of carbon chain length and degreeof saturation of fatty acids. The 1-aminoanthracene prototype addressesa problem noted from “Fast and Simple Free Fatty Acids Analysis UsingUPC²/MS” (Library Number: APTNT134753626; Part Number 720004763en) whichstates that “Reversed-phase chromatography separates lipids according toboth chain-length and degree of unsaturation. The problem lies in thefact that the dual nature of the reversed-phase separation process (adouble bond in the fatty acyl chain reduces the retention time and thefatty acyl chain length increases the retention time) can hamper theanalysis of real samples; the number of components is is often so greatthat identification becomes difficult due to coelution.” The stationaryphase of the present disclosure results in more retention forunsaturated fatty acid chains (more double bonds yield longer retentiontimes) and longer chain lengths. The separation is based on the numberof carbon atoms and the number of double bonds to increase theretention, an exceptional improvement compared to alkyl bondedstationary phases.

Those skilled in the art will understand that the present disclosure canbe used to develop and tune methods in other application areas of: finechemicals/materials (OLEDs, agrochemicals, dyes/organic dyes,conformational polymers, polymer additives and surfactants), food andenvironmental (pesticides, glycerides, edible oils, tobacco, foodadulteration), pharmaceutical/life sciences (lipid profiling, naturalproducts, DMPK/bioanalysis, impurity profiling, medicinal chemistry) andforensics/research (opiates, drugs of abuse, steroids, fatty acids,anti-depressants, gun powder components, explosives).

In addition, these materials and methods could be used as part of amultidimensional experiment (2D-SFC, SFC-LC, etc). Other materials tocombine these with include Argentation (silver impregnated materials)and existing lipid methods, such as those on CSH brand columns.

Example 10 Separation of Compounds of Interest Using Various StationaryPhases of the Present Disclosure

Additional chromatographic materials were prepared according to theprior Examples using different selectors. Lipid analysis was performedon these additional stationary phases. The materials tested includestationary phases using the following selectors: 1-Aminoanthracene,2-Picolylamine, Pyridine, 6-aminoquinoline, Aniline, Diol, and4-n-octylaniline.

The general chromatographic conditions are provided as follows: Sample:GLC85 lipids (Nu-chek Prep), 1 g/L in CHCl₃; diluted 20× with 1:1IPA:Heptane. System: UPC2 w/SQD2, ESI-ionization. Mobile phase: 97.5/2.5MeOH/H2O w/0.1M NH3 make-up flow. 3.0×50 mm columns. The samplecontained a mixture of C₄-C₂₄ lipids (m/z: 87; 115.1; 143.1; 171.1;185.1; 199.2; 213.2; 227.2; 241.2; 255.2; 269.2; 283.3; 311.3; 339.3;225.2; 239.2; 253.2; 267.2; 277.2; 279.2; 281.2; 303.2; 305.2; 307.3;309.3; 327.2; 335.3; 337.3; 365.3).

FIGS. 17-24 show the lipid separations, both profiles and per individuallipid based on carbon bond number, using these stationary phases. Ingeneral, stationary phases containing 1-Aminoanthracene and4-n-octylaniline show sufficient resolution of the mixture, as well as,an increase in retention based on the number of double bonds. Stationaryphases using 2-picolylamine, pyridine, 6-aminoquinoline and anilinecouplings and diol are unable to resolve most lipid peaks in sample.Table 9 provides a summary the performance of these stationary phases.

TABLE 9 Performance of the various Stationary Phases of the presentdisclosure Ret. Window Peak t_(R) (min) Range Material (87 to 365.3)Capacity C18:0 C18:1 C22:1 C22:6 C18 C22 Total DB 3G 1.042 21.4 1.631.67 1.84 2.06 0.1 0.22 0.43 3M 0.657 11.3 2.56 2.6 2.72 3.28 0.1 0.560.72 2F 0.622 11.3 1.35 1.39 1.47 1.67 0.08 0.2 0.32 3A 2.354 23.7 1.941.96 2.65 2.97 0.11 0.32 1.03 3E 1.674 29.6 1.85 1.8 2.18 2.14 −0.05−0.04 0.38 3F 0.731 9.5 2.23 2.26 2.39 2.87 0.1 0.48 0.64 3L 0.737 12.52.01 2.06 2.18 2.6 0.11 0.42 0.59 HSSC18SB 1.176 24.5 1.29 1.23 1.511.28 −0.11 −0.23 0.33

Table 9 provides a summary of the results. The retention window ismeasured as the time difference between the first and last eluting peakof the lipids mixture. It is desirable to maximize this value as itrepresents the separation space. Material 3A provides the highestretention window value, 200% larger than that of HSS C18 SB(comparable). The peak capacity is measured as the retention windowdivided by the average peak width of all the peaks eluted in theretention window. Material 3E provides a significant improvement overHSS C18 SB with a 20% greater peak capacity. The retention values forC18:0, C18:1, C22:1 and C22:6 represent retention times of lipids withcarbon chain lengths of 18 and 22, respectively, and 0, 1 and 6 doublebonds in the chains, respectively. Materials 3E and HSS C18 SB showdecreasing retention as the number of double bonds increases, while allothers show increasing retention. The (retention) range of all C18 andC22 lipids in the mixture are calculated by subtracting retention timeof the lipid having the highest level of unsaturation (most doublebonds), C18 or C22, respectively, from the retention time of thesaturated lipid C18 or C22, respectively. A negative value in either ofthese columns represents a decrease in retention based on the number ofdouble bonds present in the chain. The positive C18 and C22 range valuesfor materials in this list represent a unique selectivity for comparedto HSS C18 SB. The total double bond (DB) range is the total range ofC18 and C22 lipid species, calculated from the absolute value of the sumof the C18 and C22 ranges. It is desirable to maximize this value, whichis noted for material 3A. In some embodiments, the materials of thepresent disclosure provide a DB value of greater than 0.3, 0.35, 0.4,0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.1,1.2, 1.3, 1.4 or 1.5 under normal or routine chromatographic conditions.

It is observed that the best peak capacities are provided byN-octyl-aniline, HSS C18 SB (comparative column), 1-aminoanthracene andaniline based stationary phases. Peak capacity is the retention windowdivided by the average peak width at 13.4% (4 sigma width). The best C18and C22 ranges are provided by pyridine, 6-aminoquinoline,2-picolylamine and 1-aminoanthracene. The C18 range is the timedifference between C18:2 and C18:0. A negative number indicates thatC18:2 eluted before C18:0. Same for C22 range. And, the best totaldouble bond ranges are provided by 1-aminoanthracene, pyridine,6-aminoquinoline and 2-picolylamine. C18:0 has 18 carbons and 0 doublebonds; C18:1 has 18 carbons and 1 double bond; etc. Total DB (doublebond) range is the difference between the maximum and minimum retentiontimes of the C18 and C22 series.

Example 11 Separation of Vitamins D and K Using Various StationaryPhases of the Present Disclosure

Two critical pairs, Vitamins K1 and K2 and Vitamins D2 and D3, weretested using the stationary phases of the present disclosure todetermine if these materials improve the resolution of these pairs. Thechromatographic conditions are provided in Table 10. Table 11 provides asummary the results. For each of the materials tested, the resolutionsof two critical pairs, Vitamins K1 and K2 and Vitamins D2 and D3,respectively, were measured. Materials 3J, 3H and 3P significantlyimprove the resolution of the critical pairs, particularly for VitaminsK1 and K2, compared to HSS C18 SB (comparison). The percent improvementis calculated as follows:

${\%\mspace{14mu}{improvement}} = {\frac{{Rs}_{prototype} - {Rs}_{{HSS}\mspace{14mu} C\; 18\mspace{14mu}{SB}}}{{Rs}_{{HSS}\mspace{14mu} C\; 18\mspace{14mu}{SB}}} \times 100}$

It is expected that a smaller particle size of materials 3J, 3H and 3Pwould further increase the resolution of the critical pairs. Forexample, it is expected that reducing the particle size of material 3Pwould increase the observed resolution by and additional 44%. It isbelieved that the conjugated moieties of materials 3J, 3H and 3Pincrease the interactions between the chromatographic surface and thevitamins driving a unique selectivity, compared to HSS C18 SB.

In some embodiments, the materials of the present disclosure provide anRs value for Vitamins D3/D4 greater than 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,4.0 or 4.5 under normal or routine chromatographic conditions. In someembodiments, the materials of the present disclosure provide an Rs valuefor Vitamins K1/K2 greater than 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 or 4.5under normal or routine chromatographic conditions.

TABLE 10 Chromatographic Conditions Co-Solvent Methanol Gradient 3-20%co-solvent in 2 minutes Samples Vitamins: A, A acetate, E, E acetate,K1, K2, D2, D3 (0.2 mg/mL each) Column Dimension 3.0 × 50 mm Flow Rate1.2 mL/min Column Temperature 40° C. Back Pressure 2175 psi DetectorACQUITY ® PDA with SFC Flow Cell Detector Setting 235 nm 40 spec/sec(320 nm for vit. A) Weak Needle Wash iso-propanol Injection 0.5 μL (2.0μL loop with PLUNO injection mode) Instrument UPC² ® Saftware Empower

TABLE 11 Performance of the various Stationary Phases of the presentdisclosure on the separation of Vitamins D and K % improvement Vit D RsMaterial Vitamin k Rs Vitamin D Rs (compared to HSS C18 SB) HSS C18SB0.23 0.74 0 2F 2.09 0.26 −64 3L 3.40 0.46 −38 3O 2.20 0.22 −71 3N 0.300.32 −57 3F 2.77 0.43 −42 3I 3.86 0.67 −9 3G 3.54 0.54 −27 3M 2.65 / /3E 2.01 0.00 −100 3J 3.49 0.86 16 3H 3.97 0.79 7 3P 3.81 0.99 34

Unless indicated otherwise, all techniques, including the use of kitsand reagents, can be carried out according to the manufacturers'information, methods known in the art.

Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated, each individualvalue is incorporated into the specification as if it were individuallyrecited. Each of the documents cited herein (including all patents,patent applications, scientific publications, manufacturer'sspecifications, and instructions), are hereby incorporated by referencein their entirety.

The specification should be understood as disclosing and encompassingall possible permutations and combinations of the described aspects,embodiments, and examples unless the context indicates otherwise. One ofordinary skill in the art will appreciate that the invention can bepracticed by other than the summarized and described aspect,embodiments, and examples, which are presented for purposes ofillustration and not limitation.

The invention claimed is:
 1. A method of separating a compound ofinterest from a mixture, the method comprising: (a) providing themixture containing the compound of interest; (b) introducing a portionof the mixture to a chromatographic system having a chromatographiccolumn; and (c) eluting the separated compound of interest from thechromatographic column; wherein the chromatographic column has astationary phase comprising

or a mixture thereof; wherein X is a chromatographic substratecontaining silica, metal oxide, an inorganic-organic hybrid material, agroup of block copolymers, or a combination thereof.
 2. The method ofclaim 1, wherein the compound of interest is a lipid, vitamin, orpolycyclic aromatic hydrocarbon.
 3. The method of claim 2, wherein thelipid is a saturated or an unsaturated fatty acid, monoacylglyceride,diacylglyceride, triacylglyceride, phospholipid, sphingolipid orsteroid.
 4. The method of claim 2, wherein the vitamin is a watersoluble vitamin selected from the group consisting of vitamin C, vitaminB, or derivatives or combinations thereof.
 5. The method of claim 2,wherein the vitamin is a fat soluble vitamin selected from the groupconsisting of vitamin A, vitamin D, vitamin K, vitamin E, betacarotene,or derivatives or combinations thereof.
 6. The method of claim 1,wherein the chromatographic stationary phase is adapted forsupercritical fluid chromatography.
 7. The method of claim 1, whereinthe chromatographic stationary phase is adapted for carbon dioxide basedchromatography.
 8. A method for mitigating or preventing retention driftin normal phase chromatography, high-pressure liquid chromatography,solvated gas chromatography, supercritical fluid chromatography,sub-critical fluid chromatography, carbon dioxide based chromatography,hydrophilic interaction liquid chromatography, or hydrophobicinteraction liquid chromatography comprising: chromatographicallyseparating the mixture using the chromatographic system comprising thestationary phase according to claim 1, thereby mitigating or preventingretention drift.